Immunotherapy against several tumors including gastrointestinal and gastric cancer

ABSTRACT

The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated cytotoxic T cell (CTL) peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to 95 novel peptide sequences and their variants derived from HLA class I molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a § 371 National Stage Application ofPCT/EP2011/053863, filed Mar. 15, 2011, which claims priority to UnitedKingdom Application No. 1004551.6, filed Mar. 19, 2010, and U.S.Provisional Application No. 61/315,704, filed Mar. 19, 2010.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to peptides, nucleic acids and cells foruse in immunotherapeutic methods. In particular, the present inventionrelates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated epitopes recognized by CD8+ Tcells, alone or in combination with other tumor-associated peptides thatserve as active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses. The present invention relates to33 novel peptide sequences and their variants derived from HLA class Imolecules of human tumor cells that can be used in vaccine compositionsfor eliciting anti-tumor immune responses, particularly cytotoxic T cell(CTL) responses.

Description of Related Art

Gastric cancer is a disease in which malignant cells form in the liningof the stomach. Stomach or gastric cancer can develop in any part of thestomach and may spread throughout the stomach and to other organs;particularly the esophagus, lungs and the liver. Stomach cancer is thefourth most common cancer worldwide with 930,000 cases diagnosed in2002. It is a disease with a high death rate (˜800,000 per year) makingit the second most common cause of cancer death worldwide after lungcancer. It is more common in men and occurs more often in Asiancountries and in developing countries.(www.who.int/mediacentre/factsheets/fs297/en/.)

It represents roughly 2% (25,500 cases) of all new cancer cases yearlyin the United States, but it is more common in other countries. It isthe leading cancer type in Korea, with 20.8% of malignant neoplasms. InJapan gastric cancer remains the most common cancer for men. Each yearin the United States, about 13,000 men and 8,000 women are diagnosedwith stomach cancer. Most are over 70 years old.

Stomach cancer is the fourth most common cancer worldwide, after cancersof the lung, breast, and colon and rectum. Furthermore, stomach cancerremains the second most common cause of death from cancer. The AmericanCancer Society estimates that in 2007 there were an estimated onemillion new cases, nearly 70% of them in developing countries, and about800,000 deaths(www.cancer.org/downloads/STT/Global_Facts_and_Figures_2007_rev2.pdf.)

Tremendous geographic variation exists in the incidence of this diseasearound the world. Rates of the disease are highest in Asia and parts ofSouth America and lowest in North America. The highest death rates arerecorded in Chile, Japan, South America, and the former Soviet Union.

Gastric cancer is often diagnosed at an advanced stage, becausescreening is not performed in most of the world, except in Japan (and ina limited fashion in Korea) where early detection is often achieved.Thus, it continues to pose a major challenge for healthcareprofessionals. Risk factors for gastric cancer are Helicobacter pylori(H. pylori) infection, smoking, high salt intake, and other dietaryfactors. A few gastric cancers (1% to 3%) are associated with inheritedgastric cancer predisposition syndromes. E-cadherin mutations occur inapproximately 25% of families with an autosomal dominant predispositionto diffuse type gastric cancers. This subset of gastric cancer has beentermed hereditary diffuse gastric cancer.12 It may be useful to providegenetic counseling and to consider prophylactic gastrectomy in young,asymptomatic carriers of germ-line truncating

The wall of the stomach is made up of 3 layers of tissue: the mucosal(innermost) layer, the muscularis (middle) layer, and the serosal(outermost) layer. Gastric cancer begins in the cells lining the mucosallayer and spreads through the outer layers as it grows. Four types ofstandard treatment are used. Treatment for gastric cancer may involvesurgery, chemotherapy, radiation therapy or chemoradiation. Surgery isthe primary treatment for gastric cancer. The goal of surgery is toaccomplish a complete resection with negative margins (R0 resection).However, approximately 50% of patients with locoregional gastric cancercannot undergo an R0 resection. R1 indicates microscopic residual cancer(positive margins); and R2 indicates gross (macroscopic) residual cancerbut not distant disease. Patient outcome depends on the initial stage ofthe cancer at diagnosis (NCCN Clinical Practice Guidelines inOncology™).

The 5-year survival rate for curative surgical resection ranges from30-50% for patients with stage II disease and from 10-25% for patientswith stage III disease. These patients have a high likelihood of localand systemic relapse. Metastasis occurs in 80-90% of individuals withstomach cancer, with a six month survival rate of 65% in those diagnosedin early stages and less than 15% of those diagnosed in late stages.

Thus, there remains a need for new efficacious and safe treatment optionfor gastric cancer, prostate carcinoma, oral cavity carcinomas, oralsquamous carcinoma (OSCC), acute myeloid leukemia (AML), H.pylori-induced MALT lymphoma, colon carcinoma/colorectal cancer,glioblastoma, non-small-cell lung cancer (NSCLC), cervical carcinoma,human breast cancer, prostate cancer, colon cancer, pancreatic cancers,pancreatic ductal adenocarcinoma, ovarian cancer, hepatocellularcarcinoma, liver cancer, brain tumors of different phenotypes, leukemiassuch as acute lymphoblastic leukemia (ALL), lung cancer, Ewing'ssarcoma, endometrial cancer, head and neck squamous cell carcinoma,epithelial cancer of the larynx, oesophageal carcinoma, oral carcinoma,carcinoma of the urinary bladder, ovarian carcinomas, renal cellcarcinoma, atypical meningioma, papillary thyroid carcinoma, braintumors, salivary duct carcinoma, cervical cancer, extranodal T/NK-celllymphomas, Non-Hodgkins Lymphoma and malignant solid tumors of the lungand breast and other tumors enhancing the well-being of the patientswithout using chemotherapeutic agents or other agents which may lead tosevere side effects.

The present invention incorporates peptides which stimulate the immunesystem and act as anti-tumor-agents in a non-invasive fashion.

SUMMARY

Stimulation of an immune response is dependent upon the presence ofantigens recognised as foreign by the host immune system. The discoveryof the existence of tumour associated antigens has raised thepossibility of using a host's immune system to intervene in tumourgrowth.

Various mechanisms of harnessing both the humoral and cellular arms ofthe immune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognising and destroying tumour cells. The isolation ofcytotoxic T-cells (CTLs) from tumour-infiltrating cell populations orfrom peripheral blood suggests that such cells play an important role innatural immune defenses against cancer. CD8-positive T-cells (TCD8+) inparticular, which recognise Class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10amino acid residues derived from proteins or defect ribosomal products(DRIPs) located in the cytosol, play an important role in this response.The MHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

There are two classes of MHC-molecules: MHC class I molecules that canbe found on most cells having a nucleus. MHC molecules are composed ofan alpha heavy chain and beta-2-microglobulin (MHC class I receptors) oran alpha and a beta chain (MHC class II receptors), respectively. Theirthree-dimensional conformation results in a binding groove, which isused for non-covalent interaction with peptides. MHC class I presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, DRIPs and larger peptides. MHC class II moleculescan be found predominantly on professional antigen presenting cells(APCs). They primarily present peptides of exogenous or transmembraneproteins that are taken up by APCs during the course of endocytosis, andare subsequently processed. Complexes of peptide and MHC class Imolecules are recognized by CD8-positive cytotoxic T-lymphocytes bearingthe appropriate T-cell receptor (TCR), whereas complexes of peptide andMHC class II molecules are recognized by CD4-positive-helper-T cellsbearing the appropriate TCR. It is well known that the TCR, the peptideand the MHC are thereby present in a stoichiometric amount of 1:1:1.

For a peptide to elicit a cellular immune response, it must bind to anMHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-I-binding peptides are usually 8-12 amino acidresidues in length and usually contain two conserved residues(“anchors”) in their sequence that interact with the correspondingbinding groove of the MHC-molecule. In this way each MHC allele has a“binding motif” determining which peptides can bind specifically to thebinding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules being expressed bytumor cells, they also have to be recognized by T cells bearing specificT cell receptors (TCR).

The antigens that are recognized by the tumor specific CTLs, that is,their epitopes, can be molecules derived from all protein classes, suchas enzymes, receptors, transcription factors, etc. which are expressedand, as compared to unaltered cells of the same origin, up-regulated incells of the respective tumor.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members or NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose; most are found inmelanomas and normal melanocytes. Many of these melanocytelineage-related proteins are involved in the biosynthesis of melanin andare therefore not tumor specific but nevertheless are widely used forcancer immunotherapy. Examples include, but are not limited to,tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.

c) Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, Survivin, Telomerase or WT1.

d) Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not or in comparably small amountsby normal healthy tissues. It is furthermore desirable, that therespective antigen is not only present in a type of tumor, but also inhigh concentrations (i.e. copy numbers of the respective peptide percell). Tumor-specific and tumor-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumor cell due to a function e.g. in cell cycle control or suppressionof apoptosis. Additionally, downstream targets of the proteins directlycausative for a transformation may be upregulated and thus may beindirectly tumor-associated. Such indirect tumor-associated antigens mayalso be targets of a vaccination approach (Singh-Jasuja H., Emmerich N.P., Rammensee H. G., Cancer Immunol. Immunother. 2004 March; 453 (3):187-95). In both cases it is essential that epitopes are present in theamino acid sequence of the antigen, since such a peptide (“immunogenicpeptide”) that is derived from a tumor associated antigen should lead toan in vitro or in vivo T-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumorvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues.

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens being transcribed from these genes in an immune therapy. Thisis because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and immunological tolerance for thisparticular epitope needs to be absent or minimal. It is thereforeimportant to select only those peptides from over-expressed orselectively expressed proteins that are presented in connection with MHCmolecules against which a functional T cell can be found. Such afunctional T cell is defined as a T cell which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumor immunity. T-helper cell epitopes thattrigger a T-helper cell response of the TH1 type support effectorfunctions of CD8-positive killer T cells, which include cytotoxicfunctions directed against tumor cells displaying tumor-associatedpeptide/MHC complexes on their cell surfaces. In this waytumor-associated T-helper cell peptide epitopes, alone or in combinationwith other tumor-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions which stimulate anti-tumor immuneresponses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: FIG. 1A) Exemplary mass spectrum from CDC2-001demonstrating its presentation on primary tumor sample GC2464.NanoESI-LCMS was performed on a peptide pool eluted from the GC sample2464. The mass chromatogram for m/z 597.3501±0.001 Da, z=2 shows apeptide peak at the retention time 151.63 min. FIG. 1B) The detectedpeak in the mass chromatogram at 151.63 min revealed a signal of m/z597.3501 in the MS spectrum. FIG. 1C) A collisionally induced decay massspectrum from the selected precursor m/z 597.3501 recorded in thenanoESI-LCMS experiment at the given retention time confirmed thepresence of CDC2-001 in the GC2464 tumor sample. FIG. 1D) Thefragmentation pattern of the synthetic CDC2-001 reference peptide wasrecorded and compared to the generated natural TUMAP fragmentationpattern shown in C for sequence verification.

FIGS. 2A and 2B: Expression profiles of mRNA of selected proteins innormal tissues and in 25 gastric cancer samples

FIG. 2A) CDC2 (Probeset ID: 203213_at)

FIG. 2B) ASPM (Probeset ID: 219918_s_at)

FIG. 3: Exemplary results of peptide-specific in vitro immunogenicity ofclass I TUMAPs. CDS+ T cells were primed using artificial APCs loadedwith relevant (left panel) and irrelevant peptide (right panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by double staining with relevantplus irrelevant A *2402-multimers. Shown cells are gated on live CDS+lymphocytes and the numbers in the plots represent percentages ofmultimer-positive cells.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

As used herein and except as noted otherwise, all terms are defined asgiven below. The term “peptide” is used herein to designate a series ofamino acid residues, connected one to the other typically by peptidebonds between the alpha-amino and carbonyl groups of the adjacent aminoacids. The peptides are preferably 9 amino acids in length, but can beas short as 8 amino acids in length, and as long as 10, 11, 12, 13 or 14amino acids in length.

The term “oligopeptide” is used herein to designate a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.The length of the oligopeptide is not critical to the invention, as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 14 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse.

A T-cell “epitope” requires a short peptide that is bound to a class IMHC receptor, forming a ternary complex (MHC class I alpha chain,beta-2-microglobulin, and peptide) that can be recognized by a T cellbearing a matching T-cell receptor binding to the MHC/peptide complexwith appropriate affinity. Peptides binding to MHC class I molecules aretypically 8-14 amino acids in length, and most typically 9 amino acidsin length.

In humans there, are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-A*024 are examples of different MHC class I alleles that can beexpressed from these loci.

Table 1: Expression frequencies F of HLA*A024 and the most frequentHLA*A02402 serotypes. Frequencies are deduced from haplotype frequenciesGf within the American population adapted from Mori et al. (Mori et al.1017-27) employing the Hardy-Weinberg formula F=1−(1−Gf)². For detailsrefer to Chanock et al. (Chanock et al. 1211-23).

Expression frequencies of HLA*24 and A*2402 serotypes worldwide

Calculated phenotype Allele Population from Allele Frequency A*24Philippines 65% A*24 Russia Nenets 61% A*2402 Japan 59% A*24 Malaysia58% A*2402 Philippines 54% A*24 India 47% A*24 South Korea 40% A*24 SriLanka 37% A*24 China 32% A*2402 India 29% A*24 Australia West 22% A*24USA 22% A*24 Russia Samara 20% A*24 South Amerika 20% A*24 Europa 18%

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence. The term “codingregion” refers to that portion of a gene which either naturally ornormally codes for the expression product of that gene in its naturalgenomic environment, i.e., the region coding in vivo for the nativeexpression product of the gene.

The coding region can be from an non-mutated (“normal”), mutated oraltered gene, or can even be from a DNA sequence, or gene, whollysynthesized in the laboratory using methods well known to those of skillin the art of DNA synthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a T-cell response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment,” when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. This means that anysuch fragment will necessarily contain as part of its amino acidsequence a segment, fragment or portion, that is substantiallyidentical, if not exactly identical, to a sequence of SEQ ID NO: 1 to33, which correspond to the naturally occurring, or “parent” proteins ofthe SEQ ID NO: 1 to 33. When used in relation to polynucleotides, theseterms refer to the products produced by treatment of saidpolynucleotides with any of the common endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:Percent Identity=100[I−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference; and R is the number of bases or amino acids inthe Reference Sequence over the length of the alignment with theCompared Sequence with any gap created in the Reference Sequence alsobeing counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original peptides disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain, if not otherwise stated. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

Preferably, when the CTLs specific for a peptide of SEQ IDs NO: 1 to 33are tested against the substituted peptides, the peptide concentrationat which the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 pM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from more than one individual, at least two, and morepreferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

Immunotherapeutic Approaches for Treatment

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has now raised thepossibility of using a host's immune system to intervene in tumorgrowth. Various mechanisms of harnessing both the humoral and cellulararms of the immune system are currently explored for cancerimmunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofcytotoxic T-cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defenses against cancer. CD8-positive T-cells inparticular, which recognize class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 12residues derived from proteins or defect ribosomal products (DRIPS)located in the cytosols, play an important role in this response. TheMHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

MHC class I molecules can be found on most cells having a nucleus whichpresent peptides that result from proteolytic cleavage of mainlyendogenous, cytosolic or nuclear proteins, DRIPS, and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation in literature.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not by normal healthy tissues or incomparably small amounts. It is furthermore desirable, that therespective antigen is not only present in a type of tumor, but also inhigh concentrations (i.e. copy numbers of the respective peptide percell). Tumor-specific and tumor-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumor cell due to a function e.g. in cell cycle control or apoptosis.Additionally, also downstream targets of the proteins directly causativefor a transformation may be upregulated and thus be indirectlytumor-associated. Such indirectly tumor-associated antigens may also betargets of a vaccination approach. Essential is in both cases thepresence of epitopes in the amino acid sequence of the antigen, sincesuch peptide (“immunogenic peptide”) that is derived from a tumorassociated antigen should lead to an in vitro or in vivoT-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumorvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues (Lemmel et al. 450-54; Weinschenk etal. 5818-27).

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens being transcribed from these genes in an immune therapy. Thisis because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and immunological tolerance for thisparticular epitope needs to be absent or minimal. It is thereforeimportant to select only those peptides from over-expressed orselectively expressed proteins that are presented in connection with MHCmolecules against which a functional T cell can be found. Such afunctional T cell is defined as a T cell that upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumor immunity. T-helper cell epitopes thattrigger a T-helper cell response of the TH1 type support effectorfunctions of CD8-positive killer T cells, which include cytotoxicfunctions directed against tumor cells displaying tumor-associatedpeptide/MHC complexes on their cell surfaces. In this way,tumor-associated T-helper cell peptide epitopes, alone or in combinationwith other tumor-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions that stimulate anti-tumor immuneresponses.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by eitherCD8-positive CTLs (MHC class I molecule) or by CD4-positive CTLs (MHCclass II molecule) is important in the development of tumor vaccines. Itis therefore an object of the present invention, to provide compositionsof peptides that contain peptides binding to MHC complexes of eitherclass.

Considering the severe side-effects and expense associated with treatingcancer better prognosis and diagnostic methods are desperately needed.Therefore, there is a need to identify other factors representingbiomarkers for cancer in general and gastric cancer in particular.

Furthermore, there is a need to identify factors that can be used in thetreatment of cancer in general and gastric cancer in particular.

Furthermore there is no established therapeutic design for gastriccancer patients with biochemical relapse after radical prostatectomy,usually caused by residual tumor left in situ in the presence of locallyadvanced tumor growth. New therapeutic approaches that confer lowermorbidity with comparable therapeutic efficacy relative to the currentlyavailable therapeutic approaches would be desirable.

The present invention provides peptides that are useful in treatinggastric cancer and other tumors that overexpress the peptides of theinvention. These peptides were shown by mass spectrometry to benaturally presented by HLA molecules on primary human gastric cancersamples (see example 1, and FIG. 1).

The source gene from which the peptides are derived were shown to behighly overexpressed in gastric cancer, renal cell carcinoma, coloncancer, non-small cell lung carcinoma, adenocarcinoma, prostate cancer,benign neoplasm and malignant melanoma compared with normal tissues (seeexample 2, and FIG. 2) demonstrating a high degree of tumor associationof the peptide, i.e. these peptides are strongly presented on tumortissue but not on normal tissues.

HLA-bound peptides can be recognized by the immune system, specificallyby T lymphocytes/T cells. T cells can destroy the cells presenting therecognized HLA/peptide complex, e.g. gastric cancer cells presenting thederived peptides.

All peptides, that were compatible with the validation platform—seeexample 3—, of the present invention have been shown to be capable ofstimulating T cell responses (see Example 3 and FIG. 3). Thus, thepeptides are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt. As usedherein, “a pharmaceutically acceptable salt” refers to a derivative ofthe disclosed peptides wherein the peptide is modified by making acid orbase salts of the agent. For example, acid salts are prepared from thefree base (typically wherein the neutral form of the drug has a neutral—NH2 group) involving reaction with a suitable acid. Suitable acids forpreparing acid salts include both organic acids, e.g., acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, salicylicacid, and the like, as well as inorganic acids, e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and thelike. Conversely, preparation of basic salts of acid moieties which maybe present on a peptide are prepared using a pharmaceutically acceptablebase such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine or the like.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates) or hydrochloricacid (chlorides).

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from gastric cancer cells and since it was determinedthat these peptides are not present in normal tissues, these peptidescan be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies can assist apathologist in diagnosis of cancer. Detection of certain peptides bymeans of antibodies, mass spectrometry or other methods known in the artcan tell the pathologist that the tissue is malignant or inflamed orgenerally diseased. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmunosurveillance. Thus, presence of peptides shows that this mechanismis not exploited by the analyzed cells.

The peptides might be used to analyze lymphocyte responses against thosepeptides such as T cell responses or antibody responses against thepeptide or the peptide complexed to MHC molecules. These lymphocyteresponses can be used as prognostic markers for decision on furthertherapy steps. These responses can also be used as surrogate markers inimmunotherapy approaches aiming to induce lymphocyte responses bydifferent means, e.g. vaccination of protein, nucleic acids, autologousmaterials, adoptive transfer of lymphocytes. In gene therapy settings,lymphocyte responses against peptides can be considered in theassessment of side effects. Monitoring of lymphocyte responses mightalso be a valuable tool for follow-up examinations of transplantationtherapies, e.g. for the detection of graft versus host and host versusgraft diseases.

The peptides can be used to generate and develop specific antibodiesagainst MHC/peptide complexes. These can be used for therapy, targetingtoxins or radioactive substances to the diseased tissue. Another use ofthese antibodies can be targeting radionuclides to the diseased tissuefor imaging purposes such as PET. This use can help to detect smallmetastases or to determine the size and precise localization of diseasedtissues.

In addition, they can be used to verify a pathologist's diagnosis of acancer based on a biopsied sample.

Table 2 shows the peptides according to the present invention, theirrespective SEQ ID NO, and the source proteins from which these peptidesmay arise. All peptides bind the HLA A*024 alleles.

TABLE 2 SEQ ID Peptide Source NO: Code Sequence Protein(s)Peptides of the present invention  1 CDC2-001 LYQILQGIVF CDK1  2ASPM-002 SYNPLWLRI ASPM  3 UCHL5-001 NYLPFIMEL UCHL5  4 MET-006SYIDVLPEF MET  5 PROM1-001 SYIIDPLNL PROM1  6 MMP11-001 VWSDVTPLTF MMP11 7 MST1R-001 NYLLYVSNF MST1R  8 NFYB-001 VYTTSYQQI NFYB  9 SMC4-001HYKPTPLYF SMC4 10 UQCRB-001 YYNAAGFNKL UQCRB 11 PPAP2C-001 AYLVYTDRLPPAP2C 12 AVL9-001 FYISPVNKL AVL9 13 NUF2-001 VYGIRLEHF NUF2 14 ABL1-001TYGNLLDYL ABL1 15 MUC6-001 NYEETFPHI MUC6 16 ASPM-001 RYLWATVTI ASPM 17EPHA2-005 VYFSKSEQL EPHA2 18 MMP3-001 VFIFKGNQF MMP3 19 NUF2-002RFLSGIINF NUF2 20 PLK4-001 QYASRFVQL PLK4 21 ATAD2-002 KYLTVKDYL ATAD222 COL12A1-001 VYNPTPNSL COL12A1 23 COL6A3-001 SYLQAANAL COL6A3 24FANCI-001 FYQPKIQQF FANCI 25 RPS11-001 YYKNIGLGF RPS11 26 ATAD2-001AYAIIKEEL ATAD2 27 ATAD2-003 LYPEVFEKF ATAD2 28 HSP90B1-001 KYNDTFWKEFHSP90B1 29 SIAH2-001 VFDTAIAHLF SIAH2 30 SLC6A6-001 VYPNWAIGL SLC6A6 31IQGAP3-001 VYKVVGNLL IQGAP3 32 ERBB3-001 VYIEKNDKL ERBB3 33 KIF2C-001IYNGKLFDLL KIF2C Further interesting HLA A*024 peptides of the invention 34 CCDC88A- QYIDKLNEL CCDC88A 001 35 CCNB1-003MYMTVSIIDRF CCNB1 36 CCND2-001 RYLPQCSYF CCND2 37 CCNE2-001 IYAPKLQEFCCNE2 38 CEA-010 IYPDASLLI CEACAM1, CEACAM5, CEACAM6 39 CLCN3-001VYLLNSTTL CLCN3 40 DNAJC10-001 IYLEVIHNL DNAJC10 41 DNAJC10-002AYPTVKFYF 42 EIF2S3-001 IFSKIVSLF EIF2S3, LOC255308 43 EIF3L-001YYYVGFAYL EIF3L, LOC340947 44 EPPK1-001 RYLEGTSCI EPPK1 45 ERBB2-001TYLPTNASLSF ERBB2 46 GPR39-001 SYATLLHVL GPR39 47 ITGB4-001 DYTIGFGKFITGB4 48 LCN2-001 SYNVTSVLF LCN2 49 SDHC-001 SYLELVKSL LOC642502, SDHC50 PBK-001 SYQKVIELF PBK 51 POLD3-001 LYLENIDEF POLD3 52 PSMD14-001VYISSLALL PSMD14 53 PTK2-001 RYLPKGFLNQF PTK2 54 RPS11-001 YYKNIGLGFRPS11 55 TSPAN1-002 VYTTMAEHF TSPAN1 56 ZNF598-001 DYAYLREHF ZNF598 57ADAM10-001 LYIQTDHLFF ADAM10 58 MMP12-001 TYKYVDINTF MMP12 59 RRM2-001YFISHVLAF RRM2 60 TMPRSS4-001 VYTKVSAYL TMPRSS4 61 TSPAN8-001 VYKETCISFTSPAN8

In another embodiment of the invention HLA A*02 binding peptides againstgastric cancer are disclosed. For people which are A*02 and/or A*24positive, mixtures of the disclosed peptides can be used for thetreatment of gastric cancer. Preferred are mixtures of 2 to 20 peptidesand mixtures of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 and 20 peptides.

SEQ ID Peptide Source NO: Code Sequence Protein(s) 62 DIO2-001 ALYDSVILLDIO2 63 IGF2BP3-001 KIQEILTQV IGF2BP3 64 LMNB1-001 LADETLLKV LMNB1 65WNT5A-001 AMSSKFFLV WNT5A 66 FAP-003 YVYQNNIYL FAP 67 COPG-001 VLEDLEVTVCOPG, COPG2, TSGA13 68 COL6A3-002 FLLDGSANV COL6A3 69 COL6A3-003NLLDLDYEL COL6A3 70 COL6A3-004 FLIDSSEGV COL6A3 71 PSMC2-001 ALDEGDIALPSMC2 72 UBE2S-001 ALNEEAGRLLL UBE2S 73 KIF11-001 ILSPTVVSI KIF11 74ADAM8-001 KLLTEVHAA ADAM8 75 CCNB1-001 ALVQDLAKA CCNB1 76 CDC6-001ILQDRLNQV CDC6 77 F2R-001 TLDPRSFLL F2R 78 OLFM4-001 TLDDLLLYI OLFM4 79THY1-001 SLLAQNTSWLL THY1 80 CEP250-001 SLAEVNTQL CEP250 81 HIF1A-001ALDGFVMVL HIF1A 82 KRAS-001 GVDDAFYTL KRAS 83 MET-001 YVDPVITSI MET 84NCAPG-001 YLLSYIQSI NCAPG 85 NCAPG-002 QIDDVTIKI NCAPG 86 TOP-004YLYGQTTTYL TOP2A 87 TOP-005 KLDETGNSL TOP2A 88 LAMC2-002 RLDDLKMTV LAMC289 AHR-001 LTDEILTYV AHR 90 CCNB1-002 ILIDWLVQV CCNB1 91 CEACAM6-VLYGPDVPTI CEACAM6 001 92 COPB1-001 SIFGEDALANV COPB1 93 HMMR-001KLLEYIEEI HMMR 94 TPX2-001 KILEDVVGV TPX2 95 TOP-001 KIFDEILVNA TOP2A,TOP2BCell Division Cycle 2 Protein (CDC2)

The serine/threonine kinase CDC2, also known as Cdk1 (Cyclin-dependentkinase 1), plays a key role in cell cycle control. It is known as themain regulator of the G2-to-M transition. At the end of interphase, itbinds to A-type cyclins. After breakdown of the nuclear envelope, A-typecyclins are replaced by cyclin B, which forms the mitosis promotingfactor (MPF) with Cdc2. MPF is essential for driving cells throughmitosis.

The function of Cdc2 in mitosis is non-redundant and cannot becompensated by the activity of other Cdks, such as Cdk2, 4 and 6. Bycontrast, Cdc2 was reported to function in other phases of the cellcycle such as the G1-S transition as well, and it is able to substitutefor the “interphase Cdks”. Thus, Cdc2 was proposed to be the onlyessential cell cycle Cdk.

Overexpression of Cdc2 was found in several cancers, often correlatingwith poor prognosis. Among them are prostate carcinoma, oral cavitycarcinomas, oral squamous carcinoma (OSCC), acute myeloid leukemia (AML)(Qian et al.), H. pylori-induced MALT lymphoma (Banerjee et al. 217-25)and colon carcinoma (Yasui et al. 36-41). In gastric carcinoma,overexpression and/or enhanced activity has been reported and could playa causative role. Inhibitors of Cdc2 and other Cdks have been consideredas drug candidates for cancer therapy (Shapiro 1770-83).

Abnormal Spindle-Like Microcephaly Associated Protein (ASPM)

Abnormal spindle-like microcephaly associated (ASPM) is the humanorthologue of the Drosophila abnormal spindle (asp). It is involved inthe regulation of neurogenesis, and mutation causes autosomal recessiveprimary microcephaly. ASPM is localized in the spindle poles duringmitosis. ASPM overexpression was suggested as marker and potentialtherapeutic target in glioblastoma. siRNA-mediated knockdown inhibitstumor cell proliferation and neural stem cell proliferation. ASPMoverexpression may also predict enhanced invasive/metastatic potential,early tumor recurrence and poor prognosis in hepatocellular carcinoma.ASPM was upregulated in immortalized cells and non-small-cell lungcancer tissues (Jung, Choi, and Kim 703-13).

Matrix Metalloproteases 3 (MMP3)

MMP3, also called progelatinase or stromelysin 1, is an endopeptidasethat cleaves extracellular matrix (ECM) components such as fibronectin,laminin, elastin, the proteoglycan core protein and nonhelical regionsof collagens. MMPs are important in several physiological processesrequiring ECM rearrangement, such as cell migration duringembryogenesis, tissue remodeling, vascularization, involution of thelactating breast and wound healing. MMP3 also plays a role in plateletaggregation. Pathological conditions involving enhanced expression andsecretion of MMP3 include autoimmune inflammatory conditions and cancer.

MMP3 is over-expressed in some tumors, and plays a role inepithelial-mesenchymal transition (EMT). It might also contribute toearly steps in cancerogenesis, triggering epigenetic changes that resultin the generation of a malignant phenotype (Lochter et al. 180-93).Polymorphisms in the MMP3 promoter that are associated with expressionlevels were shown to impact risk and prognosis for some cancers likeesophageal adenocarcinoma (Bradbury et al. 793-98) and oral squamouscell carcinoma (Vairaktaris et al. 4095-100) (Liu et al. 430-35). H.pylori-positive gastric cancer patients with enhanced MMP3- and MMP7serum levels showed higher lymph node invasion and shorter survival. Ina cohort of 74 gastric cancer patients, MMP3 was expressed in 27% of thecases, (Murray et al. 791-97).

c-Met

c-Met mediates the potentially oncogenic activities of the hepatocyticgrowth factor (HGF)/scatter factor, including promotion of cell growth,motility, survival, extracellular matrix dissolution, and angiogenesis.Binding of HGF activates downstream signalling events including the Ras,phosphatidylinositol 3′-kinase, phospholipase Cγ, and mitogen-activatedprotein kinase-related pathways (Dong et al. 5911-18; Furge et al.10722-27; Furge, Zhang, and Vande Woude 5582-89; Montesano et al.355-65; Naldini et al. 501-04; Ponzetto et al. 4600-08). c-Met isexpressed predominantly in epithelial cells. Oncogenic activation ofc-Met (also in non-epithelial malignant tissues) can result fromamplification/over-expression, activating mutations, acquisition ofHGF/c-Met autocrine loops or constitutive phosphorylation (Di Renzo etal. 147-54; Ferracini et al. 739-49; Fischer et al. 733-39; Koochekpouret al. 5391-98; Li et al. 8125-35; Maulik et al. 41-59; Qian et al.589-96; Ramirez et al. 635-44; Tuck et al. 225-32) (Nakaigawa et al.3699-705). Constitutive activation of c-Met in HGF-over-expressingtransgenic mice promotes broad tumorigenesis (Takayama et al. 701-06;Wang et al. 1023-34). Silencing MET results in inhibition of tumorgrowth and metastasis (Corso et al. 684-93). Amplification of MET hasbeen associated with human gastric cancer progression (Lin et al.5680-89). (Yokozaki, Yasui, and Tahara 49-95).

Ubiquitin Carboxyl-Terminal Hydrolase L5 (UCHL5)

UCHL5, also known as Ubiquitin C-terminal hydrolase (UCH37) or INO80R,is a proteasome-associated deubiquitinase. It disassemblesprotein-attached poly-ubiquitin chains from the distal end by cleavingthe isopeptide bond between the C-terminal Cys76 and Lys48 (Nishio etal. 855-60). In the nucleus, UCHL5 is associated with the Ino80chromatin-remodeling complex. Upon binding of a proteasome, it becomesactivated and may contribute to the regulation of transcription or DNArepair that has been suggested to be mediated by Ino80 and theproteasome.

Ubiquitin specific proteases like UCHL5 are involved in severalprocesses such as control of cell cycle progression, differentiation,DNA replication and repair, transcription, protein quality control,immune response and apoptosis. UCHL5 might contribute to malignanttransformation. Its activity has been shown to be upregulated in humancervical carcinoma tissue as compared to adjacent normal tissue. It isable to deubiquitinate and thereby stabilize the TGF-beta receptor andits downstream mediators, the Smads, thereby enhancing TGF-betasignaling. Enhanced TGF-beta signaling can act as a tumor promoter inlate stages of cancer progression, although it has a dual function andcan also be a tumor suppressor in early stages and before initiation(Bierie and Moses 29-40; Horton et al. 138-43; Wicks et al. 8080-84;Wicks et al. 761-63).

Macrophage-Stimulating Protein Receptor (MST1R)

The MST1R (alias RON) receptor is a member of the Met family of cellsurface receptor tyrosine kinases and is primarily expressed onepithelial cells and macrophages. MST1R can induce cell migration,invasion, proliferation and survival in response to its ligand.Oncogenic properties have been shown in vitro as well as in animalmodels in vivo, and it is often deregulated in human cancers (Dussaultand Bellon, 2009). Clinical studies have shown that MST1Rover-expression is associated with poor diagnosis and metastasis. MST1Rexpression is significant in gastric carcinoma tissue and correspondingparaneoplastic tissue, but is not observed in normal gastric mucosa(Zhou et al. 236-40). Knockdown of MST1R in prostate cancer cellsresults in reduced endothelial cell chemotaxis in vitro, and in reducedtumor growth and decreased microvessel density after orthotopictransplantation into the prostate in vivo. siRNA-mediated knockdown ofMST1R in a highly tumorigenic colon cancer cell line led to reducedproliferation as compared with control cells.

Kinesin-Like Protein (KIF2C)

KIF2C is a microtubule depolymerase regulating properkinetochore-microtubule attachment during spindle formation. It isimportant for anaphase chromosome segregation and may be required tocoordinate the onset of sister centromere separation. Disturbedmicrotubule attachment at kinetochores leads to chromosomemis-segregation and aneuploidy, which is observed in most solid tumors(Maney et al. 67-131; Moore and Wordeman 537-46). KIF2C isover-expressed in breast cancer cells (Shimo et al. 62-70), coloncancer, colorectal cancer and gastric cancer (Nakamura et al. 543-49). Agastric cancer cell line (AZ521) that stably expressed KIF2C showed aincreased proliferation and migration compared to mock-transfectedcells. Elevated expression of KIF2C in gastric cancer may be associatedwith lymphatic invasion, lymph node metastasis, and poor prognosis.Treatment of breast cancer cells with small interfering RNA againstKIF2C inhibited their growth.

Structural Maintenance of Chromosomes Proteins 4 (SMC4)

SMC proteins are chromosomal ATPases that play roles in higher-orderchromosome organization and dynamics. SMC4 is a core component of thecondensin complex that plays a role in chromatin condensation and hasalso been associated with nucleolar segregation, DNA repair, andmaintenance of the chromatin scaffold. The SMC4 gene was found to beexpressed highly in normal prostate and salivary gland, very weakly incolon, pancreas, and intestine, and not at all in other tissues. RNAexpression was observed at high levels in many cancer cell lines andcancer specimens, including breast, prostate, colon and pancreaticcancer (Egland et al. 5929-34).

Ephrin Type-A Receptor 2 (EPAH2)

Eph receptors are a unique family of receptor tyrosine kinases (RTK)that play critical roles in embryonic patterning, neuronal targeting,and vascular development during normal embryogenesis. Stimulation ofEphA2 by its ligand (ephrin-A1) results in EphA2 autophosphorylation,the stimulation reverses oncogenic transformation. Eph receptors andtheir ligands, the ephrins, are frequently overexpressed in a widevariety of cancers. EphA2 is frequently overexpressed and functionallyaltered in aggressive tumor cells, and is thought to promote tumorgrowth by enhancing cell-extracellular matrix adhesion,anchorage-independent growth and angiogenesis. Overexpression of EphA2and EphrinA-1 was shown in gastric carcinoma, correlating with the depthof tumor invasion, tumor-node-metastasis (TNM) stages, lymph nodemetastasis and poor prognosis (Yuan et al. 2410-17).

ATAD2

ATAD2 (also known as ANCCA) is a new member of the AAA+ ATPase familyproteins. It enhances the transcriptional activity of androgen receptor(AR) and estrogen receptor (ER), leading to transcription of genesincluding IGF1R, IRS-2, SGK1 and surviving (AR) and cyclin D1, c-myc andE2F1 (ER), respectively. It also enhances the transcriptional activityof c-Myc. ATAD2 expression is high in several human tumors, such asbreast cancer, prostate cancer and osteosarcoma. Expression has beenassociated with poor prognosis.

AVL9

Surprisingly this protein was found as source protein, and only poor andvery limited data is available about the AVL9 protein and the functionof the corresponding gene.

Collagen Alpha-1(XII) Chain Protein (Col12A1)

Collagen alpha-1(XII) chain is a protein that in humans is encoded bythe COL12A1 gene. This gene encodes the alpha chain of type XIIcollagen, a member of the FACIT (fibril-associated collagens withinterrupted triple helices) collagen family. Type XII collagen is ahomotrimer found in association with type I collagen, an associationthat is thought to modify the interactions between collagen I fibrilsand the surrounding matrix. Alternatively spliced transcript variantsencoding different isoforms have been identified.

Collagen Alpha-3(VI) Chain Protein (COL6A3)

COL6A3 encodes the alpha-3 chain, one of the three alpha chains of typeVI collagen. The protein domains have been shown to bind extracellularmatrix proteins, an interaction that explains the importance of thiscollagen in organizing matrix components. Remodeling of theextracellular matrix through overexpression of collagen VI contributesto cisplatin resistance in ovarian cancer cells. The presence ofcollagen VI correlated with tumor grade, an ovarian cancer prognosticfactor (Sherman-Baust et al. 377-86). COL6A3 is overexpressed incolorectal tumour (Smith et al. 1452-64), salivary gland carcinoma(Leivo et al. 104-13) and differentially expressed in gastric cancer(Yang et al. 1033-40). COL6A3 was identified as one of seven genes withtumor-specific splice variants. The validated tumor-specific splicingalterations were highly consistent, enabling clear separation of normaland cancer samples and in some cases even of different tumor stages(Thorsen et al. 1214-24).

Fanconi Anemia, Complementation Group I (FANCI)

The FANCI protein localizes to chromatin in response to DNA damage andis involved in DNA repair (Smogorzewska et al. 289-301). Mutations inthe FANCI gene cause Fanconi anemia, a genetically heterogeneousrecessive disorder characterized by cytogenetic instability,hypersensitivity to DNA crosslinking agents, increased chromosomalbreakage, and defective DNA repair. Alternative splicing of FANCIresults in two transcript variants encoding different isoforms.

Heat Shock Protein 90 kDa Beta Member 1 (HSP90B1)

HSP90 (also known as glucose-regulated protein 94, Grp94), member 1 is ahuman chaperone protein. It participates in ER-associated processes:translation, protein quality control and ER-associated degradation(ERAD), ER stress sensing and calcium binding/retention of calcium inthe ER (Christianson et al. 272-82; Fu and Lee 741-44). HSP90 containsthe KDEL sequence typical for ER-retained proteins, but it also appearson the surface of tumor cells (Altmeyer et al. 340-49), as well asextracellularly. HSPs are known to be released from necrotic (but notapoptotic) cells and from cells stressed by various stimuli such as heatshock and oxidative stress, and can occur in circulation (Basu et al.1539-46; Tsan and Gao 274-79). Extracellularly, HSP90 modulates (mainlystimulates) immune responses and is involved in antigen presentation. Onthe cell surface, it may serve as receptor for pathogen entry and/orsignaling (Cabanes et al. 2827-38). In case of tumor-specific cellsurface expression or release it may induce anti-tumor immunity (Zhenget al. 6731-35). HSP90-based vaccines have been shown to immunizeagainst cancer and infectious diseases in both prophylactic andtherapeutic protocols (reviewed in (Bolhassani and Rafati 1185-99;Castelli et al. 227-33; Murshid, Gong, and Calderwood 1019-30)).

However, HSP90 can also be considered as target for tumor therapy as 1)it correlates with tumor progression and leads to resistance towardsapoptosis, also upon irradiation or chemotherapy treatment, and 2) it isoverexpressed in many tumors including GC, osteosarcoma (Guo et al.62-67), breast carcinoma (Hodorova et al. 31-35). Overexpression ofHSP90 is associated with aggressive behavior and poor prognosis in GC(Wang, Wang, and Ying 35-41; Zheng et al. 1042-49). Downregulation ofHSP90 in GC leads to apoptosis of cancer cells (Sheu, Liu, and Lane1096).

Muc 6

MUC6 is expressed in mucous cells. Its primary function is thought to bethe protection of vulnerable epithelial surfaces from damaging effectsof constant exposure to a wide range of endogenous caustic orproteolytic agents (Toribara et al., 1997). MUC6 may also play a role inepithelial organogenesis (Reid and Harris, 1999). MUC6 expression isfound in normal gastric mucosa. It is over-expressed in some cancerslike intestinal adenoma and carcinoma, pulmonary carcinoma (Hamamoto etal. 891-96), colorectal polyps (Bartman et al. 210-18), and breastcarcinoma (Pereira et al. 210-13), whereas it is not expressed in therespective normal tissues. The high expression rate of MUC6 in mucinouscarcinoma suggests was suggested to act as a barrier to cancerousextension resulting in their less aggressive biological behaviour(Matsukita et al. 26-36). MUC6 expression was lower in gastriccarcinomas than in adenomas or normal mucosa and inversely correlatedwith tumor size, depth of invasion, lymphatic and venous invasion, lymphnode metastasis and UICC staging. Down-regulation of MUC6 may contributeto malignant transformation of gastric epithelial cells and underlie themolecular bases of growth, invasion, metastasis and differentiation ofgastric carcinoma (Zheng et al. 817-23). There is also evidence thatHelicobacter pylori infection, one of the major causes of gastriccarcinoma, is associated with reduced MUC6 expression (Kang et al.29-35; Wang and Fang 425-31).

Kinetochore Protein Nuf2

NUF2 (CDCA-1) gene encodes a protein that is highly similar to yeastNuf2, a component of a conserved protein complex associated with thecentromere. Yeast Nuf2 disappears from the centromere during meioticprophase when centromeres lose their connection to the spindle polebody, and plays a regulatory role in chromosome segregation. It wasshown that survivin and hNuf2 csiRNAs temporally knockdown their mRNAscausing multinucleation and cell death by mitotic arrest, respectively(Nguyen et al. 394-403). Nuf2 and Hec1 are required for organization ofstable microtubule plus-end binding sites in the outer plate that areneeded for the sustained poleward forces required for biorientation atkinetochores (DeLuca et al. 519-31).

Nuf2 protein was found to be over-expressed in NSCLC, associated withpoor prognosis (Hayama et al. 10339-48), and in cervical cancer (Martinet al. 333-59). In surgically resected gastric cancer tissues (diffusetype, 6; intestinal type, 4), 2 variants of NUF2 were upregulated. Thealternative splicing variants detected in this study were suggested bepotentially useful as diagnostic markers and/or novel targets foranticancer therapy (Ohnuma et al. 57-68).

siRNA-mediated knockdown against NUF2 has been found to inhibit cellproliferation and induction of apoptosis in NSCLC, ovarian cancer,cervical cancer, gastric cancer, colorectal cancer and glioma (Kaneko etal. 1235-40).

Lipid Phosphate Phosphohydrolase 2 (PPAP2C)

Phosphatidic acid phosphatases (PAPs) convert phosphatidic acid todiacylglycerol, and function in de novo synthesis of glycerolipids aswell as in receptor-activated signal transduction mediated byphospholipase D. Three alternatively spliced transcript variantsencoding distinct isoforms have been reported. PPAP2C is up-regulated intransformed primary human adult mesenchymal stem cells (MSCs), andnumerous human cancers. It might be required for increased cellproliferation. Overexpression of PPAP2C, but not a catalyticallyinactive mutant, caused premature S-phase entry, accompanied bypremature cyclin A accumulation. Knockdown decreases cell proliferationby delaying entry into S phase (Flanagan et al. 249-60).

40S Ribosomal Protein S11 is a Protein (RPS11)

Ribosomes consist of a small 40S subunit and a large 60S subunit.Together these subunits are composed of 4 RNA species and approximately80 structurally distinct proteins. The RPS11 gene encodes a ribosomalprotein that is a component of the 40S subunit. RPS11 was among sixgenes found in a screen for fecal RNA-based markers for colorectalcancer diagnosis. It was specifically found in cancer-patient derivedfecal colonocytes (Yajima et al. 1029-37).

E3 Ubiquitin-Protein Ligase Seven in Absentia Homolog 2 (SIAH2)

SIAH2 is a E3 ubiquitin ligase. Among its substrates are beta-catenin,TRAF2, and DCC (deleted in colorectal cancer) (Habelhah et al. 5756-65;Hu and Fearon 724-32; Nakayama, Qi, and Ronai 443-51). SIAH2 also leadsto degradation of the nuclear protein repp86, resulting in abrogation ofthe mitotic arrest induced by overexpression of this protein(Szczepanowski et al. 485-90). SIAH2 has tumor- as well asmetastasis-promoting properties via at least two pathways, reviewed in(Nakayama, Qi, and Ronai 443-51): First, it leads to ubiquitination anddegradation of proteins in the hypoxia response pathway, which leads toenhanced transcriptional activity of hypoxia-inducible factors (HIFs)(Nakayama, Qi, and Ronai 443-51)(Calzado et al. 85-91). Second, itsuppresses Sprouty2, a specific inhibitor of Ras/ERK signaling. SIAH2activity is correlated with pancreatic tumor development likely throughits positive effect on Ras signaling (Nakayama, Qi, and Ronai 443-51).

Although the role of SIAH2 in cancer is partly controversial, somereports showing association of low levels of SIAH2 with poorer prognosisor therapy response (Confalonieri et al. 2959-68) (Jansen et al.263-71), others show a tumorigenic function (Frasor et al. 13153-57).SIAH2 inhibition has been considered as anti-cancer treatment, as it hasbeen shown to inhibit growth of xenografts in melanoma mouse models (Qiet al. 16713-18; Shah et al. 799-808), and of human lung cancer celllines engrafted into nude mice (Ahmed et al. 1606-29).

Sodium- and Chloride-Dependent Taurine Transporter (SLC6A6)

SLC6A6 is a sodium- and chloride-dependent taurine transporter (TauT)(Han et al., 2006). Taurine transporter knockout (taut−/−) mice sufferfrom chronic liver disease due to taurine deficiency, which may involvemitochondrial dysfunction (Warskulat et al., 2006). Expression of SLC6A6is repressed by the p53 tumour suppressor gene and is transactivated byproto-oncogenes such as WT1, c-Jun, and c-Myb. Over-expression of SLC6A6protects renal cells from cisplatin-induced nephrotoxicity (Han et al.,2006; Han and Chesney, 2009). SLC6A6 mRNA expression was upregulated bytumor necrosis factor alpha (TNF-alpha) in human intestinal epithelialCaco-2 cells (Mochizuki et al., 2005).

Ubiquinol-Cytochrome c Reductase Binding Protein (UQCRB)

The protein encoded by the UQCRB-gene is part of theubiquinol-cytochrome c oxidoreductase complex. It binds ubiquinone andparticipates in the transfer of electrons. Mutations in this gene areassociated with mitochondrial complex III deficiency. A pseudogene hasbeen described on the X chromosome.

The UQCRB-gene may be a potential oncogen or a tumour suppressor gene inpancreatic ductal adenocarcinoma (Harada et al. 13-24). It was found tobe overexpressed in hepatocellular carcinoma (Jia et al. 1133-39)

Human Epidermal Growth Factor Receptor 3 (ERBB3)

ERBB3 encodes a member of the epidermal growth factor receptor (EGFR)family of receptor tyrosine kinases. It is activated by neuregulins, byother ERBB and nonERBB receptors as well as by other kinases, and bynovel mechanisms. Downstream it interacts prominently with thephosphoinositol 3-kinase/AKT survival/mitogenic pathway, but also withGRB, SHC, SRC, ABL, rasGAP, SYK and the transcription regulator EBP1(Sithanandam and Anderson 413-48). ERBB3 overexpression has been foundin many cancers including gastric cancer, where it may play a keycausative role and negatively impacts prognosis (Kobayashi et al.1294-301) (Slesak et al. 2727-32). (Zhang et al. 2112-18) found thatover-expression of ERBB3 was more frequent in the diffuse type (26.2%)of gastric cancer than in the intestinal type (5.0%). In both types,overexpression was associated with poor prognosis. Approaches fortargeting of ERBB3 in cancer therapy include RNA aptamers to theextracellular domain (Chen et al. 9226-31), blockade of its geneexpression by synthetic transcription factors (Lund et al. 9082-91),small-molecule inhibitors like the vitamin E isomer γ-tocotrienol(Samant and Sylvester 563-74), miRNA (Scott et al. 1479-86) and siRNA(Sithanandam et al. 1847-59).

Prominin 1 (Prom1)

Function: Prominin-1, also called CD133, was identified as a moleculespecific for CD34+ hematopoetic progenitor cells (Yin et al., 1997) andshown to be a marker for normal stem cells and cancer stem cells (CSCs)of various tissues. It is located mainly in plasma membrane protrusions,and might be involved in the organization of membrane topology or inmaintaining the lipid composition of the plasma membrane. It wassuggested that a splice isoform of prominin-1 called AC133-2 and lackinga small exon of 27 amino acids may represent an even better stem cellmarker (Mizrak et al., 2008; Bidlingmaier et al., 2008).

Only a small percentage of tumor cells is usually positive forprominin-1, as expected for a CSC marker. Depending on the tumor type,the number of positive cells per tumor mass reaches from 1 to 15% and ismostly around 2%.

Prominin-1 has been associated with tumor formation, angiogenesis andchemoresistance (Zhu et al., 2009a) (Bruno et al., 2006; Hilbe et al.,2004) (Bertolini et al., 2009). However, prominin-1 positive cells mightbe accessible by the immune system, as they can be killed by NK cells(Castriconi et al., 2007; Pietra et al., 2009) and cytotoxic T cells(Brown et al., 2009).

While for many cancer entities it has been shown that prominin-1positive cells are functionally CSCs, and expression was frequentlyassociated with poor prognosis, there are still controversies. Somereports state that it is neither necessary nor sufficient foridentifying CSCs (Cheng et al., 2009; Wu and Wu, 2009). Possibly acombination of prominin-1 with other molecules such as CD44, or evenmultiple combinations such as prom1(+), CD34(+), CD44(+), CD38(−),CD24(−) serve as better CSC markers (Zhu et al., 2009b; Fulda andPervaiz, 2010)

In diffuse GC, PROM1 expression was suggested based on an in silicoanalysis (Katoh and Katoh, 2007) and overexpression in GC compared tonormal stomach tissue at the protein level was reported by (Smith etal., 2008). However, (Boegl and Prinz, 2009) reported that prominin-1expression was reduced in GC, especially in later stages, and claimedthat prominin-1 expression rather correlates with angiogenesis—which isalso reduced in later stages—than with tumor growth. A study using GCcell lines (Takaishi et al., 2009) claims that CD44, but not prominin-1is a CSC marker in GC.

Matrix Metalloproteinase 11 (MMP11)

Like other MMPs, MMP11 is an endopeptidase with functions in processesrequiring tissue remodeling, such as development, wound healing and scarformation. It might also negatively regulate fat homeostasis by reducingadipocyte differentiation. In contrast to other MMPs, it is not able tocleave typical extracellular matrix molecules—except collagen VI.However, other substrates have been identified such as alpha2-macroglobulin, certain serine protease inhibitors (serpins) includingalpha 1 anti-trypsin, insulin-like growth factor-binding protein-1 andthe laminin receptor. In cancer, MMP11 is mostly expressed in stromalcells surrounding tumor tissue. This has been shown for numerous tumorentities. It was stated that MMP11 is overexpressed in the stroma ofmost invasive human carcinomas, but rarely in sarcomas and othernonepithelial tumors. In most but not all cases, MMP11 is expressed instroma cells directly adjacent to the tumor, whereas the tumor cellsthemselves, normal tissues and stroma cells distant from the tumor arenegative. Higher levels of MMP11 are correlated with a malignantphenotype/higher invasiveness and bad prognosis. However, in papillarythyroid carcinomas, MMP11 expression was inversely linked to aggressivecharacteristics. MMP11 was found in tumor tissue as well as in serum ofgastric cancer patients, and expression correlated with metastasis (Yanget al.). Moreover, (Deng et al. 274-81) showed that MMP11 is highlyexpressed in tumor cell lines and primary tumor of gastric cancer—incontrast to other cancer types not exclusively in the stroma—and that itappears to enhance tumor cell proliferation.

Nuclear Transcription Factor Y Subunit Beta (NFYB)

NFYB, also called CBF-B or CBF-A is, besides NFYA and NFYC, a part ofthe heterotrimeric basal transcription factor NF-Y (also CCAAT-bindingfactor or CBF) that binds to CCAAT motifs—or the reverse motifs, ATTGG,called Y-box—in the promoters and enhancers of numerous genes. Among theNF-Y target genes are MHC class II genes, the PDGF beta-receptor,several heat shock proteins, the mismatch repair gene hMLH1, andtopoisomerase II alpha.

NFYB is not a classical oncogene, however its function might contributeto tumorigenesis. First, many cell-cycle genes such as cyclin A, cyclinB1, Aurora A and cdk1 are targets of NF-Y. Cells are arrested at G2/Mphase without functional NFYB. (Park et al.) show that upregulation ofcyclin B2 and other cell-cycle related genes in colorectaladenocarcinoma are due to NF-Y activity. Second, NF-Y activitycounteracts apoptosis. Cells lacking NF-Y undergo apoptosis due to p53activation and reduced transcription of anti-apoptotic genes containingCCAAT-boxes in their promoters, such as Bcl-2 (Benatti et al. 1415-28).Third, its tumorigenic properties are enhanced in combination with othertranscription factors. For example, mutated p53 binds to NF-Y and p300proteins, increasing the expression of NF-Y-induced cell cycle genes.

ABL1

The protein tyrosine kinase c-Abl shuttles between the nuclear andcytoplasmic compartments. Nuclear c-Abl is involved in cell growthinhibition and apoptosis, while cytoplasmic c-Abl may play a role inactin dynamics, morphogenesis and signaling induced by extracellularstimuli like growth factors and integrin ligands. Cytoplasmic c-Abl wasreported to promote mitogenesis. Activity of c-Abl protein is negativelyregulated by its SH3 domain, and deletion of the SH3 domain turns ABL1into an oncogene. In chronic myeloic leukemia (CML), the gene isactivated by translocation within the BCR (breakpoint cluster region)gene on chromosome 22. This resulting fusion protein BCR-ABL locates tothe cytosol and allows the cells to proliferate without being regulatedby cytokines (Zhao et al.). c-Abl activity is also upregulated in solidtumors, as it was shown for breast carcinomas and NSCLC. Overexpressionis not sufficient and constitutive kinase activity required proteinphosphorylation. In breast cancer cells, c-Abl phosphorylation isinduced by plasma membrane tyrosine kinases, including SFK, EGFR familymembers and the IGF-1 receptor. ABL fusion proteins have not beendetected in solid tumors (Lin and Arlinghaus, 2008). ABL was shown to beexpressed in gastric carcinoma and associated microvessels, suggesting apossible role in angiogenesis. Notably, H. pylori cytotoxin-associatedgene A (CagA) leads to activation of c-Abl, which, consequentlyphosphorylates EGFR and, thus, blocks EGFR endocytosis (Bauer, Bartfeld,and Meyer 156-69). Several tyrosine kinase inhibitors are more or lessspecific for Abl. Imatinib (Gleevec) is used as a first line therapy forCML and has also been approved for patients with advancedgastrointestinal stromal tumors (GIST), as it also targets KIT (Pytel etal. 66-76) (Croom and Perry, 2003). Other inhibitors used for cancertherapy are Dasatinib and Nilotinib (Pytel et al. 66-76) (Deremer,Ustun, and Natarajan 1956-75).

Polo-Like Kinase 4 (Plk4)

Polo kinase family members (Plk1-4) are important during cell division,regulating several steps during mitosis. Plk4 is an organizer ofcentriole formation and duplication (Rodrigues-Martins et al. 1046-50).While Plk1 is a clear oncogene, Plk4's function in cancer is ambiguous.Downregulation as well as overexpression of Plk4 has been associatedwith cancer in humans, mice and flies (Cunha-Ferreira et al. 43-49). Forinstance, in colorectal cancer, Plk4 was found overexpressed, but asmall group of patients showed strong Plk4 downregulation (Macmillan etal. 729-40). This can be explained by the fact that both overexpressionand deficiency of Plk4 lead to aberrant centriole formation, resultingin abnormal centrosome numbers and structures that are frequentlydetected in tumor cells and contribute to mitotic aberrations that causechromosome missegregation and aneuploidy (Peel et al. 834-43). (Kuriyamaet al. 2014-23). (Korzeniewski et al. 6668-75).

IQ Motif Containing GTPase Activating Protein 3 (IQGAP3)

IQGAPs participate in cellular signaling pathways as well ascytoskeletal architecture and cell adhesion. They possess a domain withsequence similarity to RasGAPs and, correspondingly, bind to smallGTPases. However (and despite their name), none of them hasGTPase-activating activity. For IQGAP1 and IQGAP2 it has been shown thatthey even stabilize the GTP-bound state of Rac1 and Cdc42, and IQGAP3was suggested to stabilize activated Ras (Nojima et al. 971-78; White,Brown, and Sacks 1817-24). Via their IQ-domain they bind tocalcium/calmodulin, and via a calponin homology domain to actinfilaments (White, Brown, and Sacks 1817-24). (Wang et al. 567-77) reportthat IQGAP3 is expressed in brain, where it associates with actinfilaments as well as Rac1 and Cdc42. It accumulates at the distal regionof axons and promotes Rac1/Ccd42-dependent axon outgrowth. The IQGAPshave been implicated in cancer. IQGAP1 is considered to be an oncogene.It enhances several cancer-related pathways like MAP kinase,beta-catenin and VEGF-mediated signaling and is overexpressed in manytumors. IQGAP2 rather seems to function as tumor suppressor and wasfound reduced in gastric cancers with poor prognosis (White, Brown, andSacks 1817-24). Little information is available about IQGAP3. (Skawranet al. 505-16) found it to be among the genes significantly upregulatedin hepatocellular carcinoma. Two studies report that IQGAP3 isspecifically expressed in proliferating (Ki67+) cells in mouse smallintestine, colon and liver (Nojima et al. 971-78) (Kunimoto et al.621-31).

Coiled-Coil Domain Containing 88a (CCDC88A)

CCDC88A is an actin-binding Akt substrate that plays a role in actinorganization and Akt-dependent cell motility in fibroblasts. TheCCDC88A/Akt pathway is also essential in VEGF-mediated postneonatalangiogenesis.

CCDC88A is also highly expressed in a variety of human malignanttissues, including breast, colon, lung, and uterine cervical carcinomas.It plays an important role in tumor progression with aberrant activationof the Akt signaling pathway.

Cyclin B1 (CCNB1)

CCNB1 is induced during G2/M phase of mitosis and forms themitosis-promoting factor (MPF) together with cyclin-dependent kinase 1(Cdk1)/Cdc2. Overexpression is found in a variety of cancers and isoften associated with poor prognosis, e.g. in breast cancer (Aaltonen etal., 2009; Agarwal et al., 2009; Suzuki et al., 2007), medulloblastoma(de et al., 2008), NSCLC (Cooper et al., 2009), cervical cancer (Zhao etal., 2006), and others. It was one of the genes included in an 11-genesignature that was found to predict short interval to disease recurrencein patients with 12 distinct types of cancer (Glinsky, 2006). Nospecific information on gastric cancer was found.

Cyclin D2 (CCND2)

CCND2 binds and activates, like other D-type cyclins (D1 and D3),cyclin-dependent kinase 4 (Cdk4) or Cdk6. This is required for G1/Stransition. CCND2 was found to be overexpressed in many tumors,including testicular and ovarian tumors (Sicinski et al., 1996),hematological malignancies (Hoglund et al., 1996; Gesk et al., 2006),and gastric cancer, where it may be caused by H. pylori infection, andassociated with poor prognosis (Yu et al., 2003). (Yu et al., 2001)(Oshimo et al., 2003) (Takano et al., 1999) (Takano et al., 2000).

Cyclin E2 (CCNE2)

CCNE2 binds and activates, like the other E-type cyclin CCNE1, Cdk2.This activity peaks at G1/S phase transition. Under healthy conditions,CCNE2 is not detectable in quiescent cells and can only be found inactively dividing tissues (Payton and Coats, 2002). It is oftenaberrantly expressed in cancer, e.g. in breast cancer, correlated to badprognosis (Desmedt et al., 2006; Ghayad et al., 2009; Payton et al.,2002; Sieuwerts et al., 2006), and in metastatic prostate cancer (Wu etal., 2009).

Carcinoembryogenic antigen-related cell adhesion molecules 1, 5 and 6(CEACAM 1, 5, and 6) CEACAMs are membrane-anchored glycoproteins thatmediate cell-cell interactions and activate integrin signaling pathways(Chan and Stanners, 2007). They may also serve as receptors forpathogens such as E. coli (Berger et al., 2004) (Hauck et al., 2006) andbe involved in immune regulation (Shao et al., 2006).

CEACAM5 and CEACAM6 have pro-cancerogenic functions. They inhibitanoikis (Ordonez et al., 2000), promote metastasis (Marshall, 2003;Ordonez et al., 2000), and disrupt cell polarization and tissuearchitecture (Chan and Stanners, 2007). The role of CEACAM1 in cancer isambiguous. It may be a tumor suppressor in early stages, and contributeto metastasis formation, tumor immune escape and angiogenesis in laterphases (Hokari et al., 2007; Liu et al., 2007; Moh and Shen, 2009). Itsfunctional role depends on the isoform, as CEACAM1 occurs in 11 splicevariants, whose ratio determines the signaling outcome (Gray-Owen andBlumberg, 2006; Leung et al., 2006; Neumaier et al., 1993; Nittka etal., 2008). The ratio of the splice variants may be altered in cancer(Gaur et al., 2008).

CEACAM5 or CEACAM6 or both are overexpressed in as many as 70% of allhuman tumors, often associated with poor prognosis (Chan and Stanners,2007; Chevinsky, 1991). Serum CEACAM5 is an established clinical markerfor colon and rectal carcinoma, high levels indicating poor prognosis orrecurrence (Chevinsky, 1991; Goldstein and Mitchell, 2005). It was alsosuggested as a marker for other entities including gastric cancer,however with limited prognostic power (Victorzon et al., 1995). CEACAM1can be up- or downregulated in cancer, depending on the entity (Kinugasaet al., 1998) (Dango et al., 2008) (Simeone et al., 2007). (Han et al.,2008) found abundant levels of CEACAM5 and CEACAM6 in nine gastriccancer cell lines, while CEACAM1 was undetectable. By contrast, ananalysis of primary tumor samples from 222 patients showed eithercytoplasmic or membranous staining for CEACAM1. The membrane-bound formwas related to enhanced angiogenesis (Zhou et al., 2009). Also the studyby (Kinugasa et al., 1998) showed an upregulation in gastricadenocarcinomas.

In some tumors, CEACAM1 is downregulated in tumor cells, which leads toupregulation of VEGF, and VEGF or hypoxic conditions may induce CEACAM1in the adjacent endothelium. Accordingly, a monoclonal antibody againstCEACAM1 blocked VEGF-induced endothelial tube formation (Oliveira-Ferreret al., 2004; Tilki et al., 2006; Ergun et al., 2000).

Especially CEACAM5 has been tested as target for anti-cancer drugs,amongst others by vaccination approaches. These studies showed thatCEACAM5 can be a target of cellular immune reactions (Cloosen et al.,2007; Marshall, 2003). An overview about CEACAM5 T cell epitopes isprovided in (Sarobe et al., 2004).

Chloride Channel 3 (CLCN3)

CLCN3 is a Cl− channel that may be volume-gated and contribute to theregulatory volume decrease (RVD) that occurs as reaction to an increasein cell volume in case of conditions like cell cycling or hypoosmosis(Lemonnier et al., 2004; Sardini et al., 2003). However, this point iscontroversially discussed (Wang et al., 2004) and the volume-reducingchannel activated during apoptosis is different from CLCN3 (Okada etal., 2006).

CLCN3 expression changes during cell cycle, peaking in S phase (Wang etal., 2004). CLCN3 currents may be important in cancer-relevant processesin entities where CLCN3 is upregulated, such as glioma: Tumor cells needto handle proliferative volume increases, encounter hypoosmoticconditions, e.g. in peritumoral edema (Ernest et al., 2005; Olsen etal., 2003; Sontheimer, 2008).

Moreover, it was reported that CLCN3 enhances etoposide resistance byincreasing acidification of the late endocytic compartment (Weylandt etal., 2007).

siRNA-mediated knockdown of CLCN3 reduced the migration ofnasopharyngeal carcinoma cells in vitro (Mao et al., 2008).

DNAJC10

DNAJC10 is a member of a supramolecular ER-associated degradation (ERAD)complex that recognizes and unfolds misfolded proteins for theirefficient retrotranslocation (Ushioda et al., 2008). The protein wasshown to be elevated in hepatocellular carcinoma (Cunnea et al., 2007).Knockdown of DNAJC10 by siRNA in neuroectodermal tumour cells increasedthe apoptotic response to the chemotherapeutic drug fenretinide(Corazzari et al., 2007). It was shown that ERdj5 decreasesneuroblastoma cell survival by down-regulating the unfolded proteinresponse (UPR) (Thomas and Spyrou, 2009).

Eukaryotic Translation Initiation Factor 2, Subunit 3 Gamma (EIF2S3)

EIF2S3 is the largest subunit of a protein complex (EIF2) recruiting theinitial methionyl-tRNA to the 40S ribosomal subunit (Clemens, 1997). Theaction of kinases that downregulate EIF activity, such as RNA-dependentprotein kinase (PKR), may be proapoptotic and tumor-suppressing (Mouniret al., 2009). In gastric cancer, higher levels of phosphorylated andunphosphorylated EIF2 were reported, and a redistribution to the nucleuswas observed. This deregulation points towards an implication ofeIF2alpha in gastrointestinal cancer (Lobo et al., 2000).

Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L)

EIF3L is one of 10-13 subunits of EIF3, which is associated with thesmall ribosomal subunit. EIF3 plays a role in prevention of prematurebinding of the large ribosomal subunit. EIF3L is among the five subunitsthat have been reported to not be essential for EIF3 formation (Masutaniet al., 2007). A screen with an antisense-library suggested thatdownregulating EIF3L enhances the anti-tumorigenic activity of5-fluorouracil in hepatocellular carcinoma cells (Doh, 2008).

Epiplakin1 (EPPK1)

EPPK1 is a plakin family gene with largely unknown functions. The plakingenes are known to function in interconnecting cytoskeletal filamentsand anchoring them at plasma membrane-associated adhesive junction(Yoshida et al., 2008).

G-Protein Coupled Receptor 39 (GPR39)

GPR39 is a Gq protein-coupled receptor that is thought to be involved ingastrointestinal and metabolic function (Yamamoto et al., 2009). Itssignalling activates cAMP and serum response elements (Hoist et al.,2004). The endogenous ligand for GPR39 is probably zinc (Chen and Zhao,2007). GPR39 is a novel inhibitor of cell death, which might represent atherapeutic target with implications for processes involving apoptosisand endoplasmic reticulum stress like cancer (Dittmer et al., 2008).GPR39 was found to be up-regulated in microarrays of both human fetalkidney HFK and blastema-enriched stem-like wilms' tumor xenografts(Metsuyanim et al., 2009), and in a hippocampal cell line resistantagainst diverse stimulators of cell death (Dittmer et al., 2008).

ERBB2/HER2/NEU

ERBB2 is a member of the EGFR family of receptor tyrosine kinases. Itsligand is not known, but it is the preferred heterodimerization partnerfor other members of the HER family (Olayioye, 2001). In carcinomas,HER2 acts as an oncogene, mainly because high-level amplification of thegene induces protein overexpression in the cellular membrane andsubsequent acquisition of advantageous properties for a malignant cell(Slamon et al., 1989). Over-expression is observed in a certainpercentage of many cancers, including gastric cancer. Mostly, it isassociated with bad prognosis (Song et al., 2010) (Yonemura et al.,1991) (Uchino et al., 1993) (Mizutani et al., 1993).

ERBB2 is the target of the monoclonal antibody trastuzumab (marketed asHerceptin), which has been suggested as treatment option for patientswith HER2-positive advanced gastric cancer, in combination withchemotherapy (Meza-Junco et al., 2009; Van Cutsem et al., 2009). Anothermonoclonal antibody, Pertuzumab, which inhibits dimerization of HER2 andHER3 receptors, is in advanced clinical trials (Kristjansdottir andDizon, 2010). The selective overexpression of HER2 and HER3 in the twohistologic types of gastric cancer (intestinal type and diffuse type) isstrongly associated with a poor prognosis (Zhang et al., 2009).

Beta-4 Integrin (ITGB4)

Integrins mediate cell adhesion as well as outside-in and inside-outsignal transduction. The integrin beta-4 subunit heterodimerizes withthe alpha-6 subunit. The resulting integrin promotes the formation ofhemidesmosomes between the intracellular keratin cytoskeleton and thebasement membrane (Giancotti, 2007). Integrin beta-4 has a dual functionin cancer, as it can mediate stable adhesion on the one hand, andpro-invasive signalling (including Ras/Erk and PI3K signalling) andangiogenesis on the other hand (Giancotti, 2007; Raymond et al., 2007).It is overexpressed in many tumors as well as in angiogenic endothelialcells, often correlating with progression and metastasis. High levelshave been in gastric cancer, particularly in stroma-invading cells(Giancotti, 2007; Tani et al., 1996). However, it was downregulated inundifferentiated-type gastric carcinoma as the tumor invaded deeper,possibly du to the gradual epithelial-mesenchymal transition, as beta-4integrin is an epithelial integrin (Yanchenko et al., 2009).

Lipocalin (LCN2)

LCN2 or neutrophil gelatinase-associated lipocalin (NGAL) is an ironregulatory protein that exists as a monomer, homodimer, or as adisulfide-linked heterodimer with MMP9 (Coles et al., 1999; Kjeldsen etal., 1993). Expression is increased in several cancers, in some casesassociated with progression. Mechanistically, it may stabilize MMP9 andalter E-cadherin-mediated cell-cell adhesion, thereby increasinginvasion. Complexes of MMP-9 and LCN2 were related with worse survivalin gastric cancer (Kubben et al., 2007) (Hu et al., 2009). Although aclear pro-tumoral effect has been observed in various tumors in humans,some studies have demonstrated that LCN2 can inhibit the pro-neoplasticfactor HIF-1alpha, FA-Kinase phosphorylation and also VEGF synthesis,thus suggesting that, in alternative conditions, LCN2 also,paradoxically, has an anti-tumoral and anti-metastatic effect inneoplasias of, for example, the colon, ovary and pancreas. (Bolignano etal., 2009; Tong et al., 2008). LCN2 may be useful for inhibiting tumorangiogenesis, in addition to suppressing tumor metastasis, in cancerswhich show ras activation (Venkatesha et al., 2006).

Succinate Dehydrogenase Complex, Subunit C (SDHC)

SDHC is one of four nuclear-encoded subunits of succinate dehydrogenase(mitochondrial complex II), which transfers electrons from succinate toubiquinone, yielding fumarate and ubiquinol. Succinate dehydrogenasedeficiency may cause GISTs (McWhinney et al., 2007). Familialgastrointestinal stromal tumors may be caused by mutations in thesubunit genes SDHB, SDHC, and SDHD, and abdominal paragangliomasassociated with gastrointestinal tumors may be caused uniquely by SDHCmutations (Pasini et al., 2008). Mutant SDHC protein in transgenic micegenerates oxidative stress and can contribute to nuclear DNA damage,mutagenesis, and ultimately, tumorigenesis (Ishii et al., 2005).Succinate dehydrogenase is considered a tumor suppressor (Baysal, 2003;Gottlieb and Tomlinson, 2005). Decreased levels of this enzyme complexmay result in tumorigenesis (Eng et al., 2003).

PDZ-Binding Kinase (PBK)

PBK is a MEK3/6-related MAPKK which activates p38 MAP kinase, e.g.downstream of growth factor receptors (Abe et al., 2000; Ayllon andO'connor, 2007). JNK may be a secondary target (Oh et al., 2007). As inadults PBK is expressed in testis (see below), a function inspermatogenesis has been proposed (Abe et al., 2000; Zhao et al., 2001).Apart from that, it contributes to proliferation and apoptosisresistance in tumor cells: It is phosphorylated and activated duringmitosis, which is necessary for spindle formation and cytokinesis(Gaudet et al., 2000; Matsumoto et al., 2004; Park et al., 2009) (Abe etal., 2007). Other growth-promoting and anti-apoptotic functions includedownregulation of p53 and histone phosphorylation (Park et al., 2006;Zykova et al., 2006) (Nandi et al., 2007). PBK has been classified ascancer-testis antigen (Abe et al., 2000; Park et al., 2006) and wasfound to be overexpressed in many cancers.

Polymerase (DNA-Directed), Delta 3, Accessory Subunit (POLD3)

The DNA polymerase delta complex is involved in DNA replication andrepair. It consists of the proliferating cell nuclear antigen (PCNA),the multisubunit replication factor C, and the 4 subunit polymerasecomplex: POLD1, POLD2, POLD3, and POLD4 (Liu and Warbrick, 2006). POLD3plays a crucial role in the efficient recycling of PCNA duringdissociation-association cycles of pol delta during elongation phase ofDNA replication (Masuda et al., 2007).

Proteasome (Prosome, Macropain) 26S Subunit, Non-ATPase, 14 (PSMD14)

PSMD14 is a component of the 26S proteasome. It belongs to the 19Scomplex (19S cap; PA700), which is responsible for substratedeubiquitination during proteasomal degradation (Spataro et al., 1997).PSMD14 overexpression in mammalian cells affects cell proliferation andthe response to cytotoxic drugs like vinblastine, cisplatin anddoxorubicin (Spataro et al., 2002). siRNA suppression of PSMD14 in HeLacells resulted in a reduction in cell viability and an increase inpolyubiquitinated protein levels (Gallery et al., 2007). Down-regulationof PSMD14 by siRNA had a considerable impact on cell viability causingcell arrest in the G0-G1 phase, ultimately leading to senescence (Byrneet al., 2010).

Proteasome (Prosome, Macropain) 26S Subunit, ATPase, 2 (PSMC2)

PSMC2 is part of the 26S proteasome system. It is a member of thetriple-A family of ATPases, which have a chaperone-like activity. Thissubunit has been shown to interact with several of the basaltranscription factors so, in addition to participation in proteasomefunctions, this subunit may participate in the regulation oftranscription. It was shown that the 26S proteasome system in skeletalmuscle can be activated by TNF-alpha (Tan et al., 2006). In HBxtransgenic mice, which bear the Hepatitis B regulatory gene HBx in theirgermline, and develop HCC, PSMC2 and other proteasome subunits areup-regulated in tumor tissues (Cui et al., 2006). The mRNA levels forthe ATPase subunit PSMC2 of the 19S complex increased in cancer cachexia(Combaret et al., 1999).

Protein Tyrosine Kinase 2 (PTK2)

PTK2 is a non-receptor tyrosine kinase which modulates integrinsignalling and may promote tumor growth, progression and metastasis((Giaginis et al., 2009); (Hauck et al., 2002); (Zhao and Guan, 2009)).PTK2 was suggested to be a marker for carcinogenesis and the progressionof cancer (Su et al., 2002; Theocharis et al., 2009; Jan et al., 2009).Overexpression and/or increased activity occurs in a wide variety ofhuman cancers including gastric cancer. PTK2 also transduces signalsdownstream of the gastrin receptor, which contributes to proliferationof gastric cancer cells (Li et al., 2008b). 8% of gastric carcinomashave been shown to carry the Epstein-Barr virus (EBV). EBV-infectedhuman gastric cancer cell line sublines presented increased PTK2phosphorylation (Kassis et al., 2002). The level of PTK2 tyrosinephosphorylation in gastric epithelial cells is reduced by thecagA-positive Helicobacter pylori product.

Tetraspanin 1 (TSPAN1) and Tetraspanin 8 (TSPAN8)

TSPAN1 and TSPAN8 belong to the family of tetraspanins which arecharacterized by four transmembrane-domains and an intracellular N- andC-terminus and which have roles in a variety of processes includingcellular adhesion, motility, activation and tumor invasion. They oftenform large molecular complexes with other proteins such as integrins atthe cell surface (Tarrant et al., 2003; Serru et al., 2000). Thefunctions of TSPAN1 are yet unknown and may include a role in secretion(Scholz et al., 2009). TSPAN1 is overexpressed in several cancers, oftencorrelating with stage, progression and worse clinical outcome. Notably,it was reported to be overexpressed in 56.98% of 86 cases of gastriccarcinoma, and overexpression correlated positively with clinical stage,infiltration and lymph node status and negatively with survival ratesand differentiation grade of the tumor (Chen et al., 2008). TSPAN8 hasbeen reported as a metastasis-associated gene in many types of tumors(PMID: 16467180). In gastrointestinal cancer, TSPAN8 expression isassociated with poor prognosis (PMID: 16849554).

Zinc Finger Protein 598 (ZNF598)

ZNF598 is a zinc finger protein with yet unknown function.

A Disintegrin and Metalloproteinase 10 (ADAM10)

ADAM10 plays a role in angiogenesis, development and tumorigenesis. Itis overexpressed in gastric carcinoma. Selective ADAM inhibitors againstADAM-10 are undergoing clinical trials for the treatment of cancer.(PMID: 19408347)

Matrix Metalloproteinase 12 (MMP12)

MMP12 is a zinc endopeptidase which degrades elastin and many othermatrix- and non-matrix-proteins and is involved in macrophage migrationand inhibition of angiogenesis (Chakraborti et al., 2003; Chandler etal., 1996; Sang, 1998). It also plays a role in pathological processesof tissue destruction like asthma, emphysema and chronic obstructivepulmonary disease (COPD), rheumatoid arthritis and tumor growth (Cataldoet al., 2003; Wallace et al., 2008). MMP12 inhibitors are discussed asagents for treatment of these conditions (Churg et al., 2007; Norman,2009). MMP12 is frequently over-expressed in cancer, where it may haveambiguous functions. While it may be involved in matrix dissolution and,thus, metastasis, it can also inhibit tumor growth through production ofangiostatin, which negatively impacts angiogenesis. Enhanced MMP12expression was reported for GC, and shown to be favorable: It negativelycorrelated with microvessel density, VEGF, tumor differentiation grade,vascular invasion, lymph node metastasis and recurrence. Patients withMMP12 over-expression demonstrated a significantly better survival rate(Cheng et al., 2010; Zhang et al., 2007b; Zhang et al., 2007a)

Ribonucleotide Reductase M2 (RRM2)

RRM2 is one of two subunits of ribonucleotide reductase, which generatesdeoxyribonucleotides from ribonucleotides. Overexpression of RRM2 hasbeen observed in tumors including gastric cancer and enhances themetastatic potential (PMID: 18941749) (PMID: 19250552) siRNA knockdownof RRM2 slowed tumor growth in various species (mouse, rat, monkey)(PMID: 17929316; PMID: 17404105).

Transmembrane Protease, Serine 4 (TMPRSS4)

TMPRSS4 is a type II transmembrane serine protease found at the cellsurface that is highly expressed in several cancer tissues, includingpancreatic, colon and gastric cancer. The biological functions ofTMPRSS4 in cancer are not yet known. TMPRSS4 has four splice variants(Scott et al., 2001; Sawasaki et al., 2004). Expression in ovariancarcinoma correlated with stage (Sawasaki et al., 2004). TMPRSS4 ishighly elevated in lung cancer tissues, and siRNA knockdown of TMPRSS4by small interfering RNA treatment in lung and colon cancer cell lineswas associated with reduction of cell invasion and cell-matrix adhesionas well as modulation of cell proliferation (Jung et al., 2008).

Deiodinase, Iodothyronine, Type II (DIO2)

DIO2 converts the prohormone thyroxine (T4) to bioactive3,3′,5-triiodothyronine (T3). It is highly expressed in the thyroid, andexpression and/or activity were found deregulated in cancers of thethyroid (de Souza Meyer et al., 2005) (Arnaldi et al., 2005). However,it was also found in other tissues, such as normal lung and lung cancer(Wawrzynska et al., 2003), and in brain tumors (Murakami et al., 2000).

Insulin-Like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3)

IGF2BP3 is primarily present in the nucleolus, where it binds IGF2 mRNAand represses its translation. It plays a role in embryogenesis and isdownregulated in adult tissues. In tumor cells it can be upregulated andis, thus, considered an oncofetal protein (Liao et al. 2005). In manycancers including gastric cancer it was found to be overexpressed,associated with poor prognosis (Jeng et al. 2009)(Jiang et al. 2006).Peptides derived from IGF2BP3 were tested in cancer vaccination studies(Kono et al. 2009).

Lamin B1 (LMNB1)

Lamin B1 is a protein of the nuclear lamina matrix and is involved innuclear stability, chromatin structure and gene expression. In earlystages of apoptosis, lamin is degraded (Neamati et al. 1995) (Sato etal. 2008b; Sato et al. 2008a; Sato et al. 2009). LMNB1 is expressed tosome extent in essentially all normal somatic cells, and preliminarystudies indicate that it may be reduced during the pathogenesis of somecancers including gastric cancer (Moss et al. 1999). In other cancers,such as hepatocellular carcinoma, LMNB1 was found upregulated andcorrelated positively with tumor stage, size and number of nodules (Limet al. 2002).

Wingless-Type MMTV Integration Site Family, Member 5A

WNT5A is a secreted signaling protein implicated in developmentalprocesses and oncogenesis. Canonical WNT5A signaling through Frizzledand LRP5/LRP6 receptors leads to maintenance of stem/progenitor cells,while non-canonical WNT5A signaling through Frizzled and ROR2/PTK/RYKreceptors controls tissue polarity, cell adhesion or movement, e.g. atthe tumor-stromal interface, leading to invasion (Katoh and Katoh,2007). It may be a tumor suppressor in some cancers, but is upregulatedin others including gastric cancer, where it contributes to progressionand metastasis and leads to poor prognosis (Li et al., 2010) (Yamamotoet al., 2009) (Kurayoshi et al., 2006).

Fibroblast Activating Protein, Alpha (FAP)

FAP is an integral membrane gelatinase. Its putative serine proteaseactivity may play a role in the control of fibroblast growth orepithelial-mesenchymal interactions during development, tissue repairand epithelial carcinogenesis (Scanlan et al. 1994). FAP has a potentialrole in cancer growth, metastasis and angiogenesis through cell adhesionand migration processes, as well as rapid degradation of ECM components.It is present on tumor cells invading the ECM, in reactivecancer-associated fibroblasts, and in endothelial cells involved inangiogenesis, but not in inactive cells of the same type. (Dolznig etal. 2005; Kennedy et al. 2009; Rettig et al. 1993; Rettig et al. 1994;Scanlan et al. 1994; Zhang et al. 2010). FAP expression has been foundin gastric cancer cells and associated stromal fibroblasts (Zhi et al.2010) (Chen et al. 2006)(Mori et al. 2004; Okada et al. 2003). In amouse model, FAP-expressing cells where shown to be a nonredundant,immune-suppressive component of the tumor microenvironment (Kraman etal. 2010). In mouse models of tumor vaccination, FAP was successfullyused as target for CD8+ and CD4+ T-cell responses (Loeffler et al. 2006;Wen et al. 2010)(Lee et al. 2005) (Fassnacht et al. 2005).

Coatomer Protein Complex, Subunit Gamma (COPG);

Coatomer Protein Complex, Subunit Gamma 2 (COPG2);

Coatomer Protein Complex, Subunit Beta 1 (COPB1)

COPG, COPG2 and COPB1 are subunits of the coatomer complex, also calledcoat protein complex 1 (COPI) that is associated with non-clathrincoated vesicles. COPI-coated vesicles mediate retrograde transport fromthe Golgi back to the ER and intra-Golgi transport (Watson et al.,2004). They may also be involved in anterograde transport (Nickel etal., 1998). The retrograde trafficking regulates, amongst others,EGF-dependent nuclear transport of EGFR, which binds to COPG (Wang etal., 2010). COPG was found to be overexpressed in lung cancer cells andlung cancer-associated microvascular endothelial cells (Park et al.,2008).

The sequence of the ubiquitously expressed COPG2 is 80% identical toGOPG (Blagitko et al., 1999). COPG2 can form a COP I-like complex inplace of GOPG, which is probably functionally redundant (Futatsumori etal., 2000).

Knockdown of COPB1 in a cystic fibrosis transmembrane conductanceregulator (CFTR) expressing cell line suggested that the coatomercomplex is involved in CRTR trafficking to the plasma membrane (Denninget al., 1992) (Bannykh et al., 2000).

Ubiquitin-Conjugating Enzyme E2S (UBE2S)

UBE2S is an auxiliary factor of the anaphase-promoting complex (APC), anE3 ubuiqitin ligase that regulates mitotic exit and G1 by targeting cellcycle regulators. UBE2S elongates ubiquitin chains after the substratesare pre-ubiquitinated by other components (Wu et al., 2010). UBE2S alsotargets the VHL protein for proteasomal degradation, thereby stabilizingHIF-1alpha (Lim et al., 2008), and possibly supporting proliferation,epithelial-mesenchymal transition, and metastasis (Chen et al., 2009)(Jung et al., 2006). UBE2S is overexpressed in several cancer entities.

Kinesin Family Member 11 (KIF11)

KIF11 is required for the assembly of a bipolar mitotic spindle. It hasbeen found upregulated in several cancers, often correlating withclinicopathological parameters (Liu et al., 2010) (Peyre et al., 2010).Small molecule inhibitors of KIF11 like S-Trityl-L-cysteine (STLC),developed as potential anti-cancer drugs, arrest cells in mitosis andpromote apoptosis of cancer cells (Tsui et al., 2009) (Wiltshire et al.,2010) (Ding et al., 2010). In the clinic, KIF11 inhibitors have shownonly modest activity (Kaan et al., 2010; Tunquist et al., 2010;Wiltshire et al., 2010; Zhang and Xu, 2008).

A Disintegrin and Metalloprotease Domain 8 (ADAM8)

ADAM8 was initially considered to be an immune-specific ADAM, but wasfound also in other cell types, often under conditions involvinginflammation and ECM remodelling, including cancers and respiratorydiseases like asthma (Koller et al. 2009). Many ADAM species, includingADAM8, are expressed in human malignant tumors, where they are involvedin the regulation of growth factor activities and integrin functions,leading to promotion of cell growth and invasion, although the precisemechanisms of these are not clear at the present time (Mochizuki andOkada 2007). In mouse gastric tumors, ADAM8 and other ADAMs wereincreased, probably due to enhanced EGFR signaling (Oshima et al. 2011).

Cell Division Cycle 6 Homolog (S. cerevisiae) (CDC6)

CDC6 is essential for the initiation of DNA replication. It localizes inthe nucleus during G1, but translocates to the cytoplasm at the start ofS phase. CDC6 also regulates replication-checkpoint activation throughinteraction with ATR (Yoshida et al. 2010). CDC6 deregulation may causethe inactivation of the INK4/ARF locus encoding three important tumorsuppressor genes: p16INK4a and p15INK4b, both activators of theretinoblastoma pathway, and ARF, an activator of p53 (Gonzalez et al.2006). siRNA knockdown of CDC6 could prevent proliferation and promoteapoptosis (Lau et al. 2006). CDC6 is upregulated in cancers includinggastric cancer (Nakamura et al. 2007) (Tsukamoto et al. 2008).

F2R Coagulation Factor II (Thrombin) Receptor (F2R)

F2R, also called proteinase activated receptor (PAR1) is a G-proteincoupled receptor. Signals by PAR1, PAR2, and PAR4 can regulate calciumrelease or mitogen-activated protein kinase activation and lead toplatelet aggregation, vascular relaxation, cell proliferation, cytokinerelease, and inflammation (Oikonomopoulou et al. 2010). F2R is thoughtto be involved in endothelial and tumor cell proliferation andangiogenesis, and is overexpressed in invasive and metastatic tumors ofmany types. The expression levels directly correlate with the degree ofinvasiveness of the cancer (Garcia-Lopez et al. 2010) (Lurje et al.2010). In gastric carcinoma cells, F2R activation can trigger an arrayof responses that promote tumor cell growth and invasion, e.g.overexpression of NF-kappaB, EGFR, and Tenascin-C (TN-C) (Fujimoto etal. 2010). Accordingly, F2R expression in gastric cancer was found to beassociated with the depth of wall invasion, peritoneal dissemination,and poor prognosis (Fujimoto et al. 2008). A mouse monoclonal anti-humanPAR1 antibody (ATAP-2), that recognizes an epitope (SFLLRNPN) within theN-terminus of the thrombin receptor, was described as well as the PAR1agonist peptide TFLLRNPNDK (Hollenberg and Compton 2002; Mari et al.1996; Xu et al. 1995)

Olfactomedin 4 (OLFM4)

OLFM4, whose function is largely unknown, is overexpressed in inflamedcolonic epithelium and a number of human tumor types, especially thoseof the digestive system (Koshida et al., 2007). OLFM4 is a robust markerfor stem cells in human intestine and marks a subset of colorectalcancer cells (van der Flier et al., 2009). OLFM4 inhibits theapoptosis-promoting protein GRIM-19 (Zhang et al., 2004) (Huang et al.,2010), regulates cell cycle and promotes S phase transition inproliferation of cancer cells. In addition, OLFM4 is associated withcancer adhesion and metastasis (Yu et al., 2011b). Forced overexpressionof OLFM4 in murine prostate tumor cells led to more rapid tumorformation in a syngeneic host (Zhang et al., 2004). OLFM4 was found tobe overexpressed in GC (Aung et al., 2006). Inhibition of OLFM4expression could induce apoptosis in the presence of cytotoxic agent ingastric cancer cells (Kim et al., 2010). Also serum OLFM4 concentrationin presurgical GC patients was enhanced as compared to healthy donors(Oue et al., 2009). OLFM4 was identified as a novel target gene forretinoic acids (RAs) and the demethylation agent 5-aza-2′-deoxycytidine.These two agents have proven to be effective in treating certain myeloidleukemia patients (Liu et al., 2010).

Thy-1 Cell Surface Antigen (THY1)

Thy-1 (CD90) is a GPI-anchored glycoprotein found on many cell typesincluding T cells, neurons, endothelial cells and fibroblasts. Thy-1 isinvolved in processes including adhesion, nerve regeneration, tumorgrowth, tumor suppression, migration, cell death, and activation of Tcells. (Rege and Hagood 2006b; Rege and Hagood 2006a) (Jurisic et al.2010). Thy-1 appears to be a marker of adult but not embryonicangiogenesis (Lee et al. 1998). Moreover, it was considered as a markerfor various kind of stem cells (mesenchymal stem cells, hepatic stemcells (“oval cells”) (Masson et al. 2006), keratinocyte stem cells(Nakamura et al. 2006) and hematopoietic stem cells (Yamazaki et al.2009)). Thy-1 is upregulated in several cancers including gastric cancerand GISTs, for which it was proposed to be a marker (Yang and Chung2008; Zhang et al. 2010) (Oikonomou et al. 2007).

Centrosomal Protein 250 kDa (CEP250)

Cep250 plays a role in the cohesion of microtubule-organizing centers(Mayor et al., 2000). It is also named centrosomal Nek2-associatedprotein or C-Nap1, as it colocalizes with and is a substrate of theserine/threonine kinase Nek2. Nek2 kinase and its substrates regulatethe linkage between centrosomes (Bahmanyar et al., 2008). At the onsetof mitosis, when centrosomes separate for bipolar spindle formation,C-Nap1 is phosphorylated and, subsequently, dissociates fromcentrosomes. In vitro experiments showed that overexpression of Cep250impaired microtubule organization at the centrosome (Mayor et al.,2002).

Hypoxia Inducible Factor 1, Alpha Subunit (Basic Helix-Loop-HelixTranscription Factor) (HIF1A)

HIF1A is the oxygen-sensitive subunit of the hypoxia-inducible factor(HIF), a transcription factor active under hypoxic conditions that arefrequently found in tumors. It mediates transcription of over 60 genesinvolved in survival, glucose metabolism, invasion, metastasis andangiogenesis (e.g. VEGF). HIF1 is overexpressed in many cancers, oftenassociated with poor prognosis, and is considered an interesting targetfor pharmacological manipulation (Griffiths et al. 2005; Quintero et al.2004; Stoeltzing et al. 2004) (Zhong et al. 1999).

In gastric cancer, HIF1A contributes to angiogenesis (Nam et al. 2011),correlates with tumor size, lower differentiation, tumor stage shortersurvival (Qiu et al. 2011) and metastasis (Wang et al. 2010) (Han et al.2006; Kim et al. 2009; Oh et al. 2008; Ru et al. 2007). It is alsothought to lead to resistance to chemotherapeutic drugs such as 5-FU viainhibition of drug-induced apoptosis and decrease of intracellular drugaccumulation (Nakamura et al. 2009) (Liu et al. 2008). TheHIF-1alpha-inhibitor 2-methoxy-estradiol significantly reducedmetastatic properties of gastric cancer cells (Rohwer et al. 2009).

v-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS)

KRAS is a member of the small GTPase superfamily and a protooncogeneinvolved in early steps of many signal transduction pathways, such asMAPK- and AKT-mediated pathways, that are potentially oncogenic. Singleamino acid substitutions lead to activating mutations, resulting in atransforming protein that plays a key role in various malignanciesincluding gastric cancer (Capella et al., 1991). Oncogenic mutations ofKRAS are infrequent in gastric cancer. In a subset of gastric cancers,the KRAS locus was amplified, resulting in overexpression of KRASprotein. Thus, gene amplification likely forms the molecular basis ofoveractivation of KRAS in gastric cancer (Mita et al., 2009). MutantKRAS alleles contribute to hypoxia-driven VEGF induction (Kikuchi etal., 2009; Zeng et al., 2010). Mutated KRAS can also be detected inserum or plasma of cancer patients and was, thus, suggested as an easilyaccessible tumor marker (Sorenson, 2000). The peptide KRAS-001 isderived from only one of two splice variants—NP_004976 (188 amino acids)and not from the splice variant—NP_203524 (189 amino acids). The splicevariants differ in their last exon, on which KRAS-001 is located.

Non-SMC Condensin I Complex, Subunit G (NCAPG)

NCAPG is part of the condensin I complex, which is composed ofstructural maintenance of chromosomes (SMC) and non-SMC proteins, andregulates chromosome condensation and segregation during mitosis(Seipold et al., 2009). NCAPG overexpression was found in numeroustumors including nasopharyngeal carcinoma (Li et al., 2010),hepatocellular carcinoma (Satow et al., 2010) and melanoma (Ryu et al.,2007). Among normal tissues, NCAPG showed highest expression in thetestis. It was suggested to be a possible proliferation marker and apotential prognostic indicator in cancer (Jager et al., 2000).

Topoisomerase (DNA) II Alpha (TOP2A) and Topoisomerase (DNA) II Beta(TOP2B)

TOP2A and TOP2B encode highly homologous isoforms of a DNAtopoisomerase, which controls and alters topologic states of DNA duringtranscription and is involved in chromosome condensation, chromatidseparation, replication and transcription. Topoisomerase is a target forseveral anticancer drugs, such as anthracyclins, and a variety ofmutations have been associated with drug resistance (Kellner et al.,2002) (Jarvinen and Liu, 2006). TOP2A (not TOP2B) is essential for cellproliferation. It is located adjacent to the HER2 oncogene and isamplified in a great majority of HER2-amplified breast tumors, but alsoin such without HER2 amplification (Jarvinen and Liu, 2003), and in manyother tumor entities. Also in a subset of gastric cancers, TOP2A wasfound amplified and overexpressed, frequently together with HER2 (Variset al., 2002) (Liang et al., 2008).

Laminin, Gamma 2 (LAMC2)

Laminins are the major non-collagenous constituents of basementmembranes. They are involved in cell adhesion, differentiation,migration, signaling, and metastasis. The gamma 2 chain together withalpha 3 and beta 3 chains constitute laminin 5. LAMC2 promotes invasivegrowth of human cancer cells in vivo. It is highly expressed by humancancers at the invasion front, and expression correlates with poorprognosis (Tsubota et al., 2010). A MMP-2-generated cleavage product oflaminin 5 is able to activate EGFR signaling and promote cell motility(Schenk et al., 2003). In gastric carcinoma, LAMC2 may be induced bymembers of the EGFR family or by Wnt5a, and invasive activity was shownto depend on LAMC2 (Tsubota et al., 2010) (Yamamoto et al., 2009).

Aryl Hydrocarbon Receptor (AHR)

AHR binds planar aromatic hydrocarbons such as TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin), and mediates transcription ofgenes including xenobiotic-metabolizing enzymes such as cytochrome P450enzymes. It also plays a role in cell cycle progression (Barhoover etal. 2010). AhR is thought to be partly associated with the tumorpromoting activity of dioxin, as it has pro-proliferative andanti-apoptotic functions, and may lead to deregulation of cell-cellcontact, dedifferentiation and enhanced motility (Watabe et al. 2010)(Dietrich and Kaina 2010) (Marlowe et al. 2008). AHR expression can bedown-regulated by TGF-beta (Dohr and Abel 1997; Wolff et al. 2001) andinduced by Wnt or beta-catenin signaling (Chesire et al. 2004). AHRoverexpression was found in many cancers including gastric cancer, whereit correlated with the frequent CYP1A1 expression (Ma et al. 2006). AHRexpression and nuclear translocation were higher in gastric cancer thanin normal tissues, and expression increased gradually duringcancerogenesis (Peng et al. 2009a). AhR pathway activation enhancesgastric cancer cell invasiveness likely through a c-Jun-dependentinduction of MMP-9 (Peng et al. 2009b). In a mouse model, expression ofa constitutively active mutant of the aryl hydrocarbon receptor (CA-AhR)results in development of stomach tumours, correlating with increasedmortality (Andersson et al. 2002; Kuznetsov et al. 2005). The functionof AhR in cancer appears to be ambiguous, as some studies also pointtowards a tumor-suppressing activity (Gluschnaider et al. 2010)(Fan etal. 2010).

Hyaluronan-Mediated Motility Receptor (RHAMM) (HMMR)

HMMR can occur on the cell surface where it binds hyaluronic acid (HA)and interacts with the HA receptor CD44. This interaction plays a rolein processes like cell motility, wound healing and invasion (Gares andPilarski, 2000). Intracellularly, HMMR associates with the cytoskeleton,microtubules, centrosomes and the mitotic spindle and plays a role incontrol of mitotic spindle integrity. HMMR is overexpressed in severalcancer tissues (Sohr and Engeland, 2008). HA was suggested to protectcancer cells against immune attack. Serum HA is often increased inmetastatic patients (Delpech et al., 1997). HMMR was identified aspromising tumor-associated antigen and possible prognostic factor in AMLand CLL. Peptides derived from HMMR have been used in anti-leukemiavaccines. HMMR-001 was tested for in vitro immunogenicity as well, butnot used for vaccination (Tzankov et al., 2011) (Greiner et al., 2010;Schmitt et al., 2008; Tabarkiewicz and Giannopoulos, 2010) (Greiner etal., 2005). HMMR overexpression was also found in several other cancers,often associated with bad prognosis. HMMR was also overexpressed ingastric cancer, often together with CD44, and was suggested tofacilitate invasion and metastasis (Li et al., 1999) (Li et al., 2000a)(Li et al., 2000b).

TPX2, Microtubule-Associated, Homolog (Xenopus laevis) (TPX2)

TPRX2 is a proliferation-associated protein expressed in S-, G(2)- andM-phases of the cell cycle and regarded as a proliferation marker(Cordes et al., 2010).

It is required for normal microtubule nucleation, e.g. for assembly ofmitotic spindles. TPX2 recruits and activates Aurora A (Bird and Hyman,2008; Moss et al., 2009). Phosphorylation of TPX2 with Polo-like kinase1 increases its ability to activate Aurora A (Eckerdt et al., 2009).TPX2 is overexpressed in many tumor types and frequentlyco-overexpressed with Aurora-A (Asteriti et al., 2010). Examples whereTPX2 overexpression was found (frequently associated with bad prognosisor later stage) are meningioma (Stuart et al., 2010), lung cancer(Kadara et al., 2009) (Lin et al., 2006; Ma et al., 2006) (Manda et al.,1999) and hepatocellular carcinoma (Shigeishi et al., 2009b) (Satow etal., 2010) (Wang et al., 2003).

The present invention therefore relates to a peptide comprising asequence that is selected from the group of SEQ ID NO: 1 to SEQ ID NO:95 or a variant thereof which is at least 80% homolog to SEQ ID NO: 1 toSEQ ID NO: 95 or a variant thereof that induces T cells cross-reactingwith said peptide, wherein said peptide is not a full-lengthpolypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 95 or avariant thereof which is at least 80% homolog to SEQ ID NO: 1 to SEQ IDNO: 95, wherein said peptide or variant has an overall length of notmore than 100, not more than 30, and most preferred from 8 to 14 aminoacids.

The present invention further relates to the peptides previouslydescribed, having the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides previouslydescribed wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 95.

The present invention further relates to the peptides previouslydescribed, wherein the peptide is modified and/or includes non-peptidebonds.

The present invention further relates to the peptides previouslydescribed, wherein the peptide is a fusion protein, in particularcomprising N-terminal amino acids of the HLA-DR antigen-associatedinvariant chain R.

The present invention further relates to a nucleic acid, encoding thepeptides previously described, provided, that the peptide is not thefull human protein.

The present invention further relates to the nucleic acid previouslydescribed which is DNA, cDNA, PNA, CNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid previously described.

The present invention further relates to a peptide as described before,a nucleic acid as described before or an expression vector as describedbefore for use in medicine.

The present invention further relates to a host cell comprising anucleic acid as described before or an expression vector as describedbefore.

The present invention further relates to the host cell described that isan antigen presenting cell.

The present invention further relates to the host cell described whereinthe antigen presenting cell is a dendritic cell.

The present invention further relates to a method of producing a peptidedescribed, the method comprising culturing the host cell described andisolating the peptide from the host cell or its culture medium.

The present invention further relates to an in vitro method forproducing activated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellfor a period of time sufficient to activate said CTL in an antigenspecific manner, wherein said antigen is any peptide described.

The present invention further relates to the method as described,wherein the antigen is loaded onto class I or II MHC molecules expressedon the surface of a suitable antigen-presenting cell by contacting asufficient amount of the antigen with an antigen-presenting cell.

The present invention further relates to the method as described,wherein the antigen-presenting cell comprises an expression vectorcapable of expressing said peptide containing SEQ ID NO 1 to SEQ ID NO33 or said variant amino acid sequence.

The present invention further relates to activated cytotoxic Tlymphocytes (CTL), produced by the method described, which selectivelyrecognise a cell which aberrantly expresses a polypeptide comprising anamino acid sequence described.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence described, the method comprisingadministering to the patient an effective number of cytotoxic Tlymphocytes (CTL) as defined.

The present invention further relates to the use of any peptidedescribed, a nucleic acid as described, an expression vector asdescribed, a cell as described, or an activated cytotoxic T lymphocyteas described as a medicament or in the manufacture of a medicament.

The present invention further relates to a use as described, wherein themedicament is a vaccine.

The present invention further relates to a use as described, wherein themedicament is active against cancer.

The present invention further relates to a use as described, whereinsaid cancer cells are gastric cancer cells, gastrointestinal,colorectal, pancreatic, lung or renal.

The present invention further relates to particular marker proteins thatcan be used in the prognosis of gastric cancer.

Further, the present invention relates to the use of these novel targetsfor cancer treatment.

As provided herein, the proteins encoded by ABL1, ADAM10, AHR, CCND2,CDC6, CDK1, CEACAM1, CEACAM5, CEACAM6, CEACAM6, COL6A3, EIF2S3,LOC255308, EPHA2, ERBB2, ERBB3, F2R, FAP, HMMR, HSP90B1, IGF2BP3, ITGB4,KIF2C, KRAS, LAMC2, LCN2, MET, MMP11, MMP12, MMP3, MST1R, NUF2, OLFM4,PROM1, RRM2, THY1, TMPRSS4, TOP2A, TSPAN1, WNT5A, HIF1A, and PTK2 weredescribed to be overexpressed in gastric cancer compared with normalgastric and other vital tissues (e.g. liver kidney, heart) inliterature.

The proteins encoded by ABL1, ADAM10, ADAMS, AHR, ASPM, ATAD2, CCDC88A,CCNB1, CCND2, CCNE2, CDC6, CDK1, CEACAM1, CEACAM5, CEACAM6, CEACAM6,CLCN3, COL6A3, EPHA2, ERBB2, ERBB3, F2R, FAP, HIF1A, HMMR, HSP90B1,IGF2BP3, IQGAP3, ITGB4, KIF11, KIF2C, KRAS, LAMC2, LCN2, MET, MMP11,MMP3, MST1R, MUC6, NCAPG, NFYB, NUF2, OLFM4, PBK, PLK4, PPAP2C, PROM1,PTK2, RRM2, SIAH2, THY1, TOP2A, TPX2, TSPAN1, TSPAN8, UBE2S, UCHL5, andWNT5A were shown to have an important role in tumorgenesis as they areinvolved in malignant transformation, cell growth, proliferation,angiogenesis or invasion into normal tissue. Also for the proteinsencoded by DNAJC10, EIF2S3, EIF3L, POLD3, PSMC2, PSMD14, and TMPRSS4,there is some evidence for cancer-relevant functions.

The proteins encoded by PROM1, WNT5A, SMC4, PPAP2C, GPR38, OLFM4 andTHY1 have been shown to be highly expressed and/or functionallyimportant in stem cells and/or cancer stem cells. PROM1 has beendiscussed as marker for gastric cancer stem cells, although data arecontroversial. Cancer stem cells are a tumor cell subpopulation withself-renewing potential required for sustained tumor growth. These cellsreside in specialized and highly organized structures, so called cancerstem cell niches that are required for the maintenance of theself-renewing potential of cancer stem cells.

Overexpression of the proteins AHR, ASPM, ATAD2, CCNB1, CCND2, CCNE2,CDK1 (CDC2), CEACAM1, CEACAM5, CEACAM6, CEACAM6, COL6A3, EPHA2, ERBB2,ERBB3, F2R, FAP, HIF1A, HMMR, HSP90B1, IGF2BP3, ITGB4, KIF11, KIF2C,KRAS, LAMC2, LCN2, LMNB1, MET, MMP11, MMP3, MST1R, MUC6, NCAPG, NUF2,OLFM4, PBK, PPAP2C, PROM1, PTK2, TMPRSS4, TPX2, TSPAN1, and WNT5A intumors has been shown to be associated with advanced disease stages andpoor prognosis for the patients.

Therefore, the present invention provides methods of identifying ananimal, preferably a human, which is likely to have gastric cancer. Inone embodiment the likelihood determined is from 80% to 100%. One suchmethod comprises determining the level of at least one of the proteinsMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 in a tumor samplefrom the animal subject. In one embodiment, the sample is obtained byradical surgery. In another embodiment, the sample is obtained by needlebiopsy.

When the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6determined is 20% or more up-regulated in cells relative to thatdetermined in benign epithelial cells of the same specimen, the animalsubject is identified as being likely to have gastric cancer.

The more different proteins of the group comprising MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB and MUC6 are up-regulated the higher thepossibility of the animal subject is identified as being likely to havegastric cancer.

In one embodiment the determination of the level of MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed in situ. In anotherembodiment the determination of the level of MST1R, UCHL5, SMC4, NFYB,PPAP2C, AVL9, UQCRB or MUC6 is performed in vitro. In still anotherembodiment, the determination of the level of MST1R, UCHL5, SMC4, NFYB,PPAP2C, AVL9, UQCRB or MUC6 is performed in vivo. In a preferredembodiment, the determination of the level of MST1R, UCHL5, SMC4, NFYB,PPAP2C, AVL9, UQCRB or MUC6 is performed by Laser Capture Microscopycoupled with a Western blot.

In a particular embodiment, the determination of the level of MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed with anantibody specific for MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB orMUC6. In another such embodiment the determination of the level ofMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed byPCR with a primer specific for an mRNA encoding MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB or MUC6. In still another embodiment thedetermination of the level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9,UQCRB or MUC6 is performed with a nucleotide probe specific for an mRNAencoding MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6. In onesuch embodiment, the determination of the level of MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed by a Northern blot. Inanother embodiment, the determination of the level of MST1R, UCHL5,SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 is performed by a ribonucleaseprotection assay. In other embodiments, immunological tests such asenzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), andWestern blots may be used to detect MST1R, UCHL5, SMC4, NFYB, PPAP2C,AVL9, UQCRB and MUC6 polypeptides in a body fluid sample (such as blood,serum, sputum, urine, or peritoneal fluid). Biopsies, tissue samples,and cell samples (such as ovaries, lymph nodes, ovarian surfaceepithelial cell scrapings, lung biopsies, liver biopsies, and any fluidsample containing cells (such as peritoneal fluid, sputum, and pleuraleffusions) may be tested by disaggregating and/or solubilizing thetissue or cell sample and subjecting it to an immunoassay forpolypeptide detection, such as ELISA, RIA, or Western blotting. Suchcell or tissue samples may also be analyzed by nucleic acid-basedmethods, e.g., reverse transcription-polymerase chain reaction (RT-PCR)amplification, Northern hybridization, or slot- or dot-blotting. Tovisualize the distribution of tumor cells within a tissue sample,diagnostic tests that preserve the tissue structure of a sample, e.g.,immunohistological staining, in situ RNA hybridization, or in situRT-PCR may be employed to detect gastric cancer marker polypeptide ormRNA, respectively. For in vivo localization of tumor masses, imagingtests such as magnetic resonance imaging (MRI) may be employed byintroducing into the subject an antibody that specifically hinds aMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide(particularly a cell surface-localized polypeptide), wherein theantibody is conjugated or otherwise coupled to a paramagnetic tracer (orother appropriate detectable moiety, depending upon the imaging methodused); alternatively, localization of an unlabeled tumor marker-specificantibody may be detected using a secondary antibody coupled to adetectable moiety.

In addition, the present invention further provides chimeric/fusionproteins/peptides comprising the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9,UQCRB or MUC6 polypeptides, and fragments thereof, including functional,proteolytic and antigenic fragments.

The fusion partner or sections of a hybrid molecule suitably provideepitopes that stimulate CD4+ T-cells. CD4+ stimulating epitopes are wellknown in the art and include those identified in tetanus toxoid. In afurther preferred embodiment the peptide is a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii). In one embodiment the peptideof the invention is a truncated human protein or a fusion protein of aprotein fragment and another polypeptide portion provided that the humanportion includes one or more inventive amino acid sequences.

Antibodies to the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6polypeptides, to the chimeric/fusion proteins comprising the MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptides, as well asto the fragments of the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB orMUC6 polypeptides, including proteolytic, and antigenic fragments, andto the chimeric/fusion proteins/peptides comprising these fragments arealso part of the present invention. In addition, methods of using suchantibodies for the prognosis of cancer, and gastric cancer inparticular, are also part of the present invention.

The antibodies of the present invention can be polyclonal antibodies,monoclonal antibodies and/or chimeric antibodies. Immortal cell linesthat produce a monoclonal antibody of the present invention are alsopart of the present invention.

One of ordinary skill in the art will understand that in some instances,higher expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB orMUC6 as a tumor marker gene will indicate a worse prognosis for asubject having gastric cancer. For example, relatively higher levelsMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 expression mayindicate a relative large primary tumor, a higher tumor burden (e.g.,more metastases), or a relatively more malignant tumor phenotype.

The more different proteins of the group comprising MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB and MUC6 are overexpressed the worse theprognosis is.

The diagnostic and prognostic methods of the invention involve usingknown methods, e.g., antibody-based methods to detect MST1R, UCHL5,SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides and nucleic acidhybridization- and/or amplification-based methods to detect MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB, and MUC6 mRNA.

In addition, since rapid tumor cell destruction often results inautoantibody generation, the gastric cancer tumor markers of theinvention may be used in serological assays (e.g., an ELISA test of asubject's serum) to detect autoantibodies against MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB or MUC6 in a subject. MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB, and MUC6 polypeptide-specific autoantibodylevels that are at least about 3-fold higher (and preferably at least5-fold or 7-fold higher, most preferably at least 10-fold or 20-foldhigher) than in a control sample are indicative of gastric cancer.

Cell-surface localized, intracellular, and secreted MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptides may all be employed foranalysis of biopsies, e.g., tissue or cell samples (including cellsobtained from liquid samples such as peritoneal cavity fluid) toidentify a tissue or cell biopsy as containing gastric cancer cells. Abiopsy may be analyzed as an intact tissue or as a whole-cell sample, orthe tissue or cell sample may be disaggregated and/or solubilized asnecessary for the particular type of diagnostic test to be used. Forexample, biopsies or samples may be subjected to whole-tissue orwhole-cell analysis of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB andMUC6 polypeptide or mRNA levels in situ, e.g., usingimmunohistochemistry, in situ mRNA hybridization, or in situ RT-PCR. Theskilled artisan will know how to process tissues or cells for analysisof polypeptide or mRNA levels using immunological methods such as ELISA,immunoblotting, or equivalent methods, or analysis of mRNA levels bynucleic acid-based analytical methods such as RT-PCR, Northernhybridization, or slot- or dot-blotting.

Kits for Measuring Expression Levels of MST1R, UCHL5, SMC4, NFYB,PPAP2C, AVL9, UQCRB, and MUC6.

The present invention provides kits for detecting an increasedexpression level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB andMUC6 as a gastric cancer marker gene in a subject. A kit for detectinggastric cancer marker polypeptide preferably contains an antibody thatspecifically binds a chosen gastric cancer marker polypeptide. A kit fordetecting gastric cancer marker mRNA preferably contains one or morenucleic acids (e.g., one or more oligonucleotide primers or probes, DNAprobes, RNA probes, or templates for generating RNA probes) thatspecifically hybridize with MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9,UQCRB, and MUC6 mRNA.

Particularly, the antibody-based kit can be used to detect the presenceof, and/or measure the level of, a MST1R, UCHL5, SMC4, NFYB, PPAP2C,AVL9, UQCRB and MUC6 polypeptide that is specifically bound by theantibody or an immunoreactive fragment thereof. The kit can include anantibody reactive with the antigen and a reagent for detecting areaction of the antibody with the antigen. Such a kit can be an ELISAkit and can contain a control (e.g., a specified amount of a particulargastric cancer marker polypeptide), primary and secondary antibodieswhen appropriate, and any other necessary reagents such as detectablemoieties, enzyme substrates and color reagents as described above. Thediagnostic kit can, alternatively, be an immunoblot kit generallycomprising the components and reagents described herein.

A nucleic acid-based kit can be used to detect and/or measure theexpression level of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB andMUC6 by detecting and/or measuring the amount of MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB and MUC6 mRNA in a sample, such as a tissue orcell biopsy. For example, an RT-PCR kit for detection of elevatedexpression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6preferably contains oligonucleotide primers sufficient to performreverse transcription of gastric cancer marker mRNA to cDNA and PCRamplification of gastric cancer marker cDNA, and will preferably alsocontain control PCR template molecules and primers to performappropriate negative and positive controls, and internal controls forquantization. One of ordinary skill in the art will understand how toselect the appropriate primers to perform the reverse transcription andPCR reactions, and the appropriate control reactions to be performed.Such guidance is found, for example, in F. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997.Numerous variations of RT-PCR are known in the art. Targeted Delivery ofimmunotoxins to MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6can be employed as therapeutic targets for the treatment or preventionof gastric cancer. For example, an antibody molecule that specificallybinds a cell surface-localized MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9,UQCRB and MUC6 polypeptide can be conjugated to a radioisotope or othertoxic compound. Antibody conjugates are administered to the subject sothat the binding of the antibody to its cognate gastric cancerpolypeptide results in the targeted delivery of the therapeutic compoundto gastric cancer cells, thereby treating an ovarian cancer.

The therapeutic moiety can be a toxin, radioisotope, drug, chemical, ora protein (see, e.g., Bera et al. “Pharmacokinetics and antitumoractivity of a bivalent disulfide-stabilized Fv immunotoxin with improvedantigen binding to erbB2” Cancer Res. 59:4018-4022 (1999)). For example,the antibody can be linked or conjugated to a bacterial toxin (e.g.,diptheria toxin, pseudomonas exotoxin A, cholera toxin) or plant toxin(e.g., ricin toxin) for targeted delivery of the toxin to a cellexpressing MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 Thisimmunotoxin can be delivered to a cell and upon binding the cellsurface-localized gastric cancer marker polypeptide, the toxinconjugated to the gastric cancer marker-specific antibody will bedelivered to the cell.

In addition, for any MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB andMUC6 polypeptide for which there is a specific ligand (e.g., a ligandthat binds a cell surface-localized protein), the ligand can be used inplace of an antibody to target a toxic compound to a gastric cancercell, as described above.

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules and humanizedversions of immunoglobulin molecules, so long as they exhibit any of thedesired properties (e.g., specific binding of an gastric cancer markerpolypeptide, delivery of a toxin to an gastric cancer cell expressing angastric cancer marker gene at an increased level, and/or inhibiting theactivity of an gastric cancer marker polypeptide) described herein.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length gastric cancer marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques. For example, a cDNA encoding a MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide, or afragment thereof, can be expressed in prokaryotic cells (e.g., bacteria)or eukaryotic cells (e.g., yeast, insect, or mammalian cells), afterwhich the recombinant protein can be purified and used to generate amonoclonal or polyclonal antibody preparation that specifically bind thegastric cancer marker polypeptide used to generate the antibody.

One of skill in the art will know that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988). For example, the antibodies may be tested in ELISA assays,Western blots, immunohistochemical staining of formalin-fixed gastriccancers or frozen tissue sections. After their initial in vitrocharacterization, antibodies intended for therapeutic or in vivodiagnostic use are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate hostanimal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fe fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the nonmodified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody for treating gastric cancer, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of gastric cancer in a subject receiving treatment may bemonitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofgastric cancer.

Because the proteins ABL1, ADAM10, AHR, CCND2, CDC6, CDK1, CEACAM1,CEACAM5, CEACAM6, CEACAM6, COL6A3, EIF2S3, LOC255308, EPHA2, ERBB2,ERBB3, F2R, FAP, HMMR, HSP90B1, IGF2BP3, ITGB4, KIF2C, KRAS, LAMC2,LCN2, MET, MMP11, MMP12, MMP3, MST1R, NUF2, OLFM4, PROM1, RRM2, THY1,TMPRSS4, TOP2A, TSPAN1, WNT5A, HIF1A, and PTK2 have been shown to behighly expressed in at least a subset of gastric cancer tissues ascompared to normal tissues, inhibition of their expression or activitymay be integrated into any therapeutic strategy for treating orpreventing gastric cancer.

The principle of antisense therapy is based on the hypothesis thatsequence-specific suppression of gene expression (via transcription ortranslation) may be achieved by intra-cellular hybridization betweengenomic DNA or mRNA and a complementary antisense species. The formationof such a hybrid nucleic acid duplex interferes with transcription ofthe target tumor antigen-encoding genomic DNA, orprocessing/transport/translation and/or stability of the target tumorantigen mRNA.

Antisense nucleic acids can be delivered by a variety of approaches. Forexample, antisense oligonucleotides or anti-sense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into tumor cells. Alternatively, viral or plasmidvectors that encode antisense RNA (or RNA fragments) can be introducedinto cells in vivo. Antisense effects can also be induced by sensesequences; however, the extent of phenotypic changes is highly variable.Phenotypic changes induced by effective antisense therapy are assessedaccording to changes in, e.g., target mRNA levels, target proteinlevels, and/or target protein activity levels.

In a specific example, inhibition of gastric tumor marker function byantisense gene therapy may be accomplished by direct administration ofantisense gastric tumor marker RNA to a subject. The antisense tumormarker RNA may be produced and isolated by any standard technique, butis most readily produced by in vitro transcription using an antisensetumor marker cDNA under the control of a high efficiency promoter (e.g.,the T7 promoter). Administration of anti-sense tumor marker RNA to cellscan be carried out by any of the methods for direct nucleic acidadministration described below.

An alternative strategy for inhibiting MST1R, UCHL5, SMC4, NFYB, PPAP2C,AVL9, UQCRB or MUC6 function using gene therapy involves intracellularexpression of an anti-MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB orMUC6 antibody or a portion of an anti-MST1R, UCHL5, SMC4, NFYB, PPAP2C,AVL9, UQCRB or MUC6 antibody. For example, the gene (or gene fragment)encoding a monoclonal antibody that specifically binds to a MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB or MUC6 polypeptide and inhibitsits biological activity is placed under the transcriptional control of aspecific (e.g., tissue- or tumor-specific) gene regulatory sequence,within a nucleic acid expression vector. The vector is then administeredto the subject such that it is taken up by gastric cancer cells or othercells, which then secrete the anti-MST1R, UCHL5, SMC4, NFYB, PPAP2C,AVL9, UQCRB or MUC6 antibody and thereby block biological activity ofthe MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 polypeptide.Preferably, the MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6polypeptides are present at the extracellular surface of gastric cancercells.

In the methods described above, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for inhibition ofgastric tumor marker protein expression. The vector can be acommercially available preparation, such as an adenovirus vector(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of thenucleic acid or vector to cells can be via a variety of mechanisms. Asone example, delivery can be via a liposome, using commerciallyavailable liposome preparations such as LIPOFECTIN, LIPOFECTAMINE(GIBCO-25 BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden,Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as wellas other liposomes developed according to procedures standard in theart. In addition, the nucleic acid or vector of this invention can bedelivered in vivo by electroporation, the technology for which isavailable from Genetronics, Inc. (San Diego, Calif.) as well as by meansof a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system that can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells antisense nucleic acid thatinhibits expression of MST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB orMUC6. The exact method of introducing the altered nucleic acid intomammalian cells is, of course, not limited to the use of retroviralvectors. Other techniques are widely available for this procedureincluding the use of adenoviral vectors, adeno-associated viral (AAV)vectors, lentiviral vectors, pseudotyped retroviral vectors. Physicaltransduction techniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms. This invention canbe used in conjunction with any of these or other commonly used genetransfer methods.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as111In, 99Tc, 14C, 131I, 3H, 32 P or 35 S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moreMST1R, UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB, and MUC6 targets and theaffinity value (Kd) is less than 1×10 μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect the MST1R,UCHL5, SMC4, NFYB, PPAP2C, AVL9, UQCRB and MUC6 proteins express insitu.

The present invention thus provides a peptide comprising a sequence thatis selected from the group of SEQ ID NO: 1 to SEQ ID NO: 95 or a variantthereof which is 85%, preferably 90% and more preferred 96%, homologousto SEQ ID NO: 1 to SEQ ID NO: 95 or a variant thereof that will induce Tcells cross-reacting with said peptide.

The peptides of the invention have the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-I.

In the present invention, the term “homologous” refers to the degree ofidentity between sequences of two amino acid sequences, i.e. peptide orpolypeptide sequences. The aforementioned “homology” is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The sequences to be compared herein may havean addition or deletion (for example, gap and the like) in the optimumalignment of the two sequences. Such a sequence homology can becalculated by creating an alignment using, for example, the ClustalWalgorithm. Commonly available sequence analysis software, morespecifically, Vector NTI, GENETYX or analysis tools provided by publicdatabases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Fong et al. 8809-14); (Appay et al. 1805-14;Colombetti et al. 2730-38; Zaremba et al. 4570-77).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in SEQ ID NO: 1 to 33. For example, a peptide may bemodified so that it at least maintains, if not improves, the ability tointeract with and bind to the binding groove of a suitable MHC molecule,such as HLA-A*02 or -DR, and in that way it at least maintains, if notimproves, the ability to bind to the TCR of activated CTL.

These CTL can subsequently cross-react with cells and kill cells thatexpress a polypeptide which contains the natural amino acid sequence ofthe cognate peptide as defined in the aspects of the invention. As canbe derived from the scientific literature (Rammensee, Bachmann, andStevanovic) and databases (Rammensee et al. 213-19), certain positionsof HLA binding peptides are typically anchor residues forming a coresequence fitting to the binding motif of the HLA receptor, which isdefined by polar, electrophysical, hydrophobic and spatial properties ofthe polypeptide chains constituting the binding groove. Thus one skilledin the art would be able to modify the amino acid sequences set forth inSEQ ID NO: 1 to 95, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated CTL, which cansubsequently cross-react with—and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

Those amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith another amino acid whose incorporation does not substantiallyaffect T-cell reactivity and does not eliminate binding to the relevantMHC. Thus, apart from the proviso given, the peptide of the inventionmay be any peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 3 Variants and motif of the peptides according to SEQ ID NO: 1 to 33 Position 1 2 3 4 5 6 7 8 9  10 CDC2-001Peptide Code (SEQ ID NO: 1) L Y Q I L Q G I V F SEQ ID NO: 98 Variants FL I F L F I ASPM-002 Peptide Code (SEQ ID NO: 2) S Y N P L W L R ISEQ ID NO: 99 Variants F L F F L F F UCHL5-001 Peptide Code(SEQ ID NO: 3) N Y L P F I M E L SEQ ID NO: 100 Variants F F I F F F IMET-006 Peptide Code (SEQ ID NO: 4) S Y I D V L P E F SEQ ID NO: 101Variants F L I F F F I PROM-001 Peptide Code (SEQ ID NO: 5) S Y I I D PL N L SEQ ID NO: 102 Variants F F I F F F I MMP11-001 Peptide Code(SEQ ID NO: 6) V W S D V T P L T F SEQ ID NO: 103 Variants Y F L I Y L YI F L F I MST1R-001 Peptide Code (SEQ ID NO: 7) N Y L L Y V S N FSEQ ID NO: 104 Variants F L I F L F I NFYB-001 Peptide Code(SEQ ID NO: 8) V Y T T S Y Q Q I SEQ ID NO: 105 Variants F L F F L F FSMC4-001 Peptide Code (SEQ ID NO: 9) H Y K P T P L Y F SEQ ID NO: 106Variants F L I F L F I UQCRB-001 Peptide Code (SEQ ID NO: 10) Y Y N A AG F N K  L SEQ ID NO: 107 Variants F F I F F F I PPAP2C-001 Peptide Code(SEQ ID NO: 11) A Y L V Y T D R L SEQ ID NO: 108 Variants F F I F F F IAVL9-001 Peptide Code (SEQ ID NO: 12) F Y I S P V N K L SEQ ID NO: 109Variants F F I F F F I NUF2-001 Peptide Code (SEQ ID NO: 13) V Y G I R LE H F SEQ ID NO: 110 Variants F L I F L F I ABL1-001 Peptide Code(SEQ ID NO: 14) T Y G N L L D Y L SEQ ID NO: 111 Variants F F I F F F IMUC-006 Peptide Code (SEQ ID NO: 15) N Y E E T F P H I SEQ ID NO: 112Variants F F L F F F L ASPM-001 Peptide Code (SEQ ID NO: 16) R Y L W A TV T I SEQ ID NO: 113 Variants F F L F F F L EPHA2-005 Peptide Code(SEQ ID NO: 17) V Y F S K S E Q L SEQ ID NO: 114 Variants F F I F F F IMMP3-001 Peptide Code (SEQ ID NO: 18) V F I F K G N Q F SEQ ID NO: 115Variants Y L I Y L Y I NUF2-002 Peptide Code (SEQ ID NO: 19) R F L S G II N F SEQ ID NO: 116 Variants Y L I Y L Y I PLK4-001 Peptide Code(SEQ ID NO: 20) Q Y A S R F V Q L SEQ ID NO: 117 Variants F F I F F F IATAD2-002 Peptide Code (SEQ ID NO: 21) K Y L T V K D Y L SEQ ID NO: 118Variants F F I F F F I COL12A1-001 Peptide Code (SEQ ID NO: 22) V Y N PT P N S L SEQ ID NO: 119 Variants F F I F F F I COL6A3-001 Peptide Code(SEQ ID NO: 23) S Y L Q A A N A L SEQ ID NO: 120 Variants F F I F F F IFANCI-001 Peptide Code (SEQ ID NO: 24) F Y Q P K I Q Q F SEQ ID NO: 121Variants F L I F L F I RSP11-001 Peptide Code (SEQ ID NO: 25) Y Y K N IG L G F SEQ ID NO: 122 Variants F L I F L F I ATAD2-001 Peptide Code(SEQ ID NO: 26) A Y A I I K E E L SEQ ID NO: 123 Variants F F I F F F IATAD2-003 Peptide Code (SEQ ID NO: 27) L Y P E V F E K F SEQ ID NO: 124Variants F L I F L F I HSP90B1-001 Peptide Code (SEQ ID NO: 28) K Y N DT F W K E F SEQ ID NO: 125 Variants F L I F L F I SIAH2-001 Peptide Code(SEQ ID NO: 29) V F D T A I A H L F SEQ ID NO: 126 Variants Y L I Y L YI SLC6A6-001 Peptide Code (SEQ ID NO: 30) V Y P N W A I G LSEQ ID NO: 127 Variants F F I F F F I IQGAP3-001 Peptide Code(SEQ ID NO: 31) V Y K V V G N L L SEQ ID NO: 128 Variants F F I F F F IERBB3-001 Peptide Code (SEQ ID NO: 32) V Y I E K N D K L SEQ ID NO: 129Variants F F I F F F I KIF2C-001 Peptide Code (SEQ ID NO: 33) I Y N G KL F D L L SEQ ID NO: 130 Variants F F I F F F I

Longer peptides may also be suitable. It is also possible, that MHCclass I epitopes, although usually from 8 to 11 amino acids long, aregenerated by peptide processing from longer peptides or proteins thatinclude the actual epitope. It is preferred that the residues that flankthe actual epitope are residues that do not substantially affectproteolytic cleavage necessary to expose the actual epitope duringprocessing.

Accordingly, the present invention also provides peptides and variantsof MHC class I epitopes wherein the peptide or variant has an overalllength of not more than 100, not more than 30, and most preferred from 8to 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I. Binding of a peptide or avariant to a MHC complex may be tested by methods known in the art.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 95.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 95 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is afusion protein which comprises, for example, the 80 N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (p33, in thefollowing “Ii”) as derived from the NCBI, GenBank Accession numberX00497.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCbinding and T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides, which contain NH—CO bonds instead of CO—NHpeptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH2-NH, —CH2S—, —CH2CH2-, —CH═CH—,—COCH2-, —CH(OH)CH2-, and —CH2SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH2-NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH3.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals.

Woodward's Reagent K may be used to modify specific glutamic acidresidues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be usedto form intra-molecular crosslinks between a lysine residue and aglutamic acid residue.

For example, diethylpyrocarbonate is a reagent for the modification ofhistidyl residues in proteins. Histidine can also be modified using4-hydroxy-2-nonenal.

The reaction of lysine residues and other α-amino groups is, forexample, useful in binding of peptides to surfaces or the cross-linkingof proteins/peptides. Lysine is the site of attachment ofpoly(ethylene)glycol and the major site of modification in theglycosylation of proteins.

Methionine residues in proteins can be modified with e.g. iodoacetamide,bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lu et al (1981)and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is done using 20% piperidine inN, N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethandithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example (Bruckdorfer, Marder, and Albericio 29-43) and thereferences as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilisation of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, CNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc, New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by (Saikiet al. 487-91)). This method may be used for introducing the DNA into asuitable vector, for example by engineering in suitable restrictionsites, or it may be used to modify the DNA in other useful ways as isknown in the art. If viral vectors are used, pox- or adenovirus vectorsare preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063,4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the preprotrypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209 (No ATCC 31343).Preferred eukaryotic host cells include yeast, insect and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic and colon cell lines. Yeast host cellsinclude YPH499, YPH500 and YPH501, which are generally available fromStratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferredmammalian host cells include Chinese hamster ovary (CHO) cells availablefrom the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 availablefrom the ATCC as CRL 1658, monkey kidney-derived COS-1 cells availablefrom the ATCC as CRL 1650 and 293 cells which are human embryonic kidneycells. Preferred insect cells are Sf9 cells which can be transfectedwith baculovirus expression vectors. An overview regarding the choice ofsuitable host cells for expression can be found in, for example, thetextbook of Paulina Balbás and Argelia Lorence “Methods in MolecularBiology Recombinant Gene Expression, Reviews and Protocols,” Part One,Second Edition, ISBN 978-1-58829-262-9, and other literature known tothe person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110, and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Transformation of yeast cells is described in Sherman et al (1986)Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y.The method of Beggs (1978) Nature 275, 104-109 is also useful. Withregard to vertebrate cells, reagents useful in transfecting such cells,for example calcium phosphate and DEAE-dextran or liposome formulations,are available from Stratagene Cloning Systems, or Life TechnologiesInc., Gaithersburg, Md. 20877, USA. Electroporation is also useful fortransforming and/or transfecting cells and is well known in the art fortransforming yeast cell, bacterial cells, insect cells and vertebratecells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) are currently under investigation for the treatment of prostatecancer (Sipuleucel-T) (Rini et al. 67-74; Small et al. 3089-94).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Brunsvig et al. 1553-64; Staehler et al.).

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre et al 1985.

Preferably, the host cell before transfection expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the costimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive CTLs.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 95 or a variant aminoacid sequence thereof.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al (1995) and Kawakami etal (1992) use autologous tumor-infiltrating lymphocytes in thegeneration of CTL. Plebanski et al (1995) makes use of autologousperipheral blood lymphocytes (PLBs) in the preparation of CTL. Jochmuset al (1997) describes the production of autologous CTL by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus. Hill et al (1995) and Jerome et al (1993) make use ofB cells in the production of autologous CTL. In addition, macrophagespulsed with peptide or polypeptide, or infected with recombinant virus,may be used in the preparation of autologous CTL. S. Walter et al. 2003describe the in vitro priming of T cells by using artificial antigenpresenting cells (aAPCs), which is also a suitable way for generating Tcells against the peptide of choice. In this study, aAPCs were generatedby the coupling of preformed MHC:peptide complexes to the surface ofpolystyrene particles (microbeads) by biotin:streptavidin biochemistry.This system permits the exact control of the MHC density on aAPCs, whichallows to selectively elicit high- or low-avidity antigen-specific Tcell responses with high efficiency from blood samples. Apart fromMHC:peptide complexes, aAPCs should carry other proteins withco-stimulatory activity like anti-CD28 antibodies coupled to theirsurface. Furthermore such aAPC-based systems often require the additionof appropriate soluble factors, e. g. cytokines like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al (1994)) which describes the development of cowpea mosaicvirus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to 95.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease which can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass I) and/or stromal cells surrounding the tumor (tumor cells) (whichsometimes also express MHC class I; (Dengjel et al. 4163-70)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art and can be found, e.g. in (Dudley et al. 850-54; Dudley etal. 2346-57; Rosenberg et al. 889-97; Rosenberg et al. 1676-80; Yee etal. 16168-73); reviewed in (Gattinoni et al. 383-93) and (Morgan etal.).

Any molecule of the invention, i.e. the peptide, nucleic acid,expression vector, cell, activated CTL, T-cell receptor or the nucleicacid encoding it is useful for the treatment of disorders, characterizedby cells escaping an immune response. Therefore any molecule of thepresent invention may be used as medicament or in the manufacture of amedicament. The molecule may be used by itself or combined with othermolecule(s) of the invention or (a) known molecule(s).

Preferably, the medicament of the present invention is a vaccine. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker 1993). The peptide mayalso be tagged, may be a fusion protein, or may be a hybrid molecule.The peptides whose sequence is given in the present invention areexpected to stimulate CD4 or CD8 T cells. However, stimulation of CD8CTLs is more efficient in the presence of help provided by CD4 T-helpercells. Thus, for MHC Class I epitopes that stimulate CD8 CTL the fusionpartner or sections of a hybrid molecule suitably provide epitopes whichstimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes arewell known in the art and include those identified in the presentinvention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth in SEQ ID NO:1 to 33 and at least oneadditional peptide, preferably two to 50, more preferably two to 25,even more preferably two to 15 and most preferably two, three, four,five, six, seven, eight, nine, ten, eleven, twelve or thirteen peptides.The peptide(s) may be derived from one or more specific TAAs and maybind to MHC class I molecules.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be DNA, cDNA, PNA, CNA,RNA or a combination thereof. Methods for designing and introducing sucha nucleic acid are well known in the art. An overview is provided bye.g. (Pascolo et al. 117-22). Polynucleotide vaccines are easy toprepare, but the mode of action of these vectors in inducing an immuneresponse is not fully understood. Suitable vectors and delivery systemsinclude viral DNA and/or RNA, such as systems based on adenovirus,vaccinia virus, retroviruses, herpes virus, adeno-associated virus orhybrids containing elements of more than one virus. Non-viral deliverysystems include cationic lipids and cationic polymers and are well knownin the art of DNA delivery. Physical delivery, such as via a “gene-gun,”may also be used. The peptide or peptides encoded by the nucleic acidmay be a fusion protein, for example with an epitope that stimulates Tcells for the respective opposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CTLs and helper-T(TH) cells to an antigen, and would thus be considered useful in themedicament of the present invention. Suitable adjuvants include, but arenot limited to, 1018 ISS, aluminium salts, AMPLIVAX®, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived fromflagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA®),resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21,Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch,ISS, ISCOMATRIX® (saponin-based adjuvant), ISCOMs, JuvImmune, LipoVac,MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK® (denileukin diftitox),OspA, PepTel® vector system, poly(lactid co-glycolid) [PLG]-based anddextran microparticles, talactoferrin SRL172, Virosomes and otherVirus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys,Aquila's QS21 STIMULON®, which is derived from saponin, mycobacterialextracts and synthetic bacterial cell wall mimics, and other proprietaryadjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such asFreund's or GM-CSF are preferred. Several immunological adjuvants (e.g.,MF59) specific for dendritic cells and their preparation have beendescribed previously (Allison and Krummel 932-33). Also cytokines may beused. Several cytokines have been directly linked to influencingdendritic cell migration to lymphoid tissues (e.g., TNF-), acceleratingthe□ maturation of dendritic cells into efficient antigen-presentingcells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha. IFN-beta) [Gabrilovich 1996].

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg 471-84). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AMPLIGEN® (Rintatolimod),HILTONOL® (poly-(ICLC)), poly(IC-R), poly(I:C12U), non-CpG bacterial DNAor RNA as well as immunoactive small molecules and antibodies such ascyclophosphamide, sunitinib, Bevacizumab, CELEBREX®, NCX-4016,sildenafil, tadalafil, vardenafil, sorafenib, temozolomide,temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171,anti-CTLA4, other antibodies targeting key structures of the immunesystem (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan without undue experimentation.

Preferred adjuvants are imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides andderivates, poly-(I:C) and derivates, RNA, sildenafil, and particulateformulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), imiquimod, resiquimod, andinterferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), immiquimod and resimiquimod.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is imiquimod or resiquimod.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavours, lubricants, etc.The peptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed. 2000, AmericanPharmaceutical Association and pharmaceutical press. The composition canbe used for a prevention, prophylaxis and/or therapy of adenomatous orcancerous diseases. Exemplary formulations can be found in EP2113253.

The present invention provides a medicament that useful in treatingcancer, in particular gastric cancer, renal cell carcinoma, coloncancer, non-small cell lung carcinoma, adenocarcinoma, prostate cancer,benign neoplasm and malignant melanoma.

The present invention further includes a kit comprising:

(a) a container that contains a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contains instructions on or associated with thecontainer that indicates directions for reconstitution and/or use. Forexample, the label may indicate that the lyophilized formulation is toreconstituted to peptide concentrations as described above. The labelmay further indicate that the formulation is useful or intended forsubcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, a anti-angiogenesis agent orinhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceuticalcomposition thereof. The components of the kit may be pre-complexed oreach component may be in a separate distinct container prior toadministration to a patient. The components of the kit may be providedin one or more liquid solutions, preferably, an aqueous solution, morepreferably, a sterile aqueous solution. The components of the kit mayalso be provided as solids, which may be converted into liquids byaddition of suitable solvents, which are preferably provided in anotherdistinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably the administration is s.c., and most preferably,i.d. Administration may be by infusion pump.

Since the peptides of the invention derived from MST1R, UCHL5, SMC4,NFYB, PPAP2C, AVL9, UQCRB and MUC6 were isolated from gastric cancer,the medicament of the invention is preferably used to treat gastriccancer.

The present invention will now be described in the following examplesthat describe preferred embodiments thereof, nevertheless, without beinglimited thereto. For the purposes of the present invention, allreferences as cited herein are incorporated by reference in theirentireties.

EXAMPLES Example 1

Identification of Tumor Associated Peptides Presented on Cell Surface

Tissue Samples

Patients' tumor tissues were provided by Kyoto Prefectural University ofMedicine (KPUM), Kyoto, Japan, Osaka City University Graduate School ofMedicine (OCU), Osaka, Japan, and University Hospital Tübingen, Germany.Written informed consents of all patients had been given before surgery.Tissues were shock-frozen in liquid nitrogen immediately after surgeryand stored until isolation of TUMAPs at −80° C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K. 1991; Seeger, F. H. T 1999} using the HLA-A, -B,-C-specific antibody W6/32, the HLA-A*02-specific antibody BB7.2,CNBr-activated sepharose, acid treatment, and ultrafiltration.

Methods

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in anLTQ-ORBITRAP® hybrid mass spectrometer (ThermoFisher Scientific)equipped with an ESI source. Peptide pools were loaded directly onto theanalytical fused-silica micro-capillary column (75 μm i.d.×250 mm)packed with 1.7 μm C18 reversed-phase material (Waters) applying a flowrate of 400 nL per minute. Subsequently, the peptides were separatedusing a two-step 180 minute-binary gradient from 10% to 33% B at a flowrate of 300 nL per minute. The gradient was composed of Solvent A (0.1%formic acid in water) and solvent B (0.1% formic acid in acetonitrile).A gold coated glass capillary (PicoTip, New Objective) was used forintroduction into the nanoESI source. The LTQ-ORBITRAP® massspectrometer was operated in the data-dependent mode using a TOPSstrategy. In brief, a scan cycle was initiated with a full scan of highmass accuracy in the ORBITRAP® mass spectrometer (R=30 000), which wasfollowed by MS/MS scans also in the orbitrap (R=7500) on the 5 mostabundant precursor ions with dynamic exclusion of previously selectedions. Tandem mass spectra were interpreted by SEQUEST and additionalmanual control. The identified peptide sequence was assured bycomparison of the generated natural peptide fragmentation pattern withthe fragmentation pattern of a synthetic sequence-identical referencepeptide. FIG. 1 shows an exemplary spectrum obtained from tumor tissuefor the MHC class I associated peptide CDC2-001 and its elution profileon the UPLC system.

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Not all peptides identified as being presented on the surface of tumorcells by MHC molecules are suitable for immunotherapy, because themajority of these peptides are derived from normal cellular proteinsexpressed by many cell types. Only few of these peptides aretumor-associated and likely able to induce T cells with a highspecificity of recognition for the tumor from which they were derived.In order to identify such peptides and minimize the risk forautoimmunity induced by vaccination the inventors focused on thosepeptides that are derived from proteins that are over-expressed on tumorcells compared to the majority of normal tissues.

The ideal peptide will be derived from a protein that is unique to thetumor and not present in any other tissue. To identify peptides that arederived from genes with an expression profile similar to the ideal onethe identified peptides were assigned to the proteins and genes,respectively, from which they were derived and expression profiles ofthese genes were generated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by different clinicalsites (see Example 1) after written informed consent had been obtainedfrom each patient. Tumor tissue specimens were snap-frozen in liquidnitrogen immediately after surgery and later homogenized with mortar andpestle under liquid nitrogen. Total RNA was prepared from these samplesusing TRI Reagent (Ambion, Darmstadt, Germany) followed by a cleanupwith RNeasy (QIAGEN, Hilden, Germany); both methods were performedaccording to the manufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted. Leukocytes were isolated fromblood samples of 4 healthy volunteers.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalisation, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0.

The expression profiles of source genes of the present invention thatare highly over-expressed in gastric cancer are shown in FIG. 2.

Example 3

In Vitro Immunogenicity for IMA941 MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, we performed investigations using awell established in vitro stimulation platform already described by(Walter, S, Herrgen, L, Schoor, O, Jung, G, Wernet, D, Buhring, H J,Rammensee, H G, and Stevanovic, S; 2003, Cutting edge: predeterminedavidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coatedmicrospheres, J. Immunol., 171, 4974-4978). With this system we couldshow positive immunogenicity (i. e. expansion of specific T cells)results for 47 of 54 tested HLA-A*2402 restricted TUMAPs and for 3 of 3tested HLA-A*0201 restricted TUMAPs of the invention, demonstrating thatthese peptides are T-cell epitopes against which CD8+ precursor T cellsexist in humans (Table 4).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells (aAPC) loaded with peptide-MHC complex (pMHC) andanti-CD28 antibody, we first isolated CD8 T cells from fresh HLA-A*24leukapheresis products or from HLA-A*2 buffy coats of healthy donorsobtained from the Blood Bank Tuebingen.

CD8 T cells were either directly enriched or PBMCs (peripheral bloodmononuclear cells) were isolated first by using standard gradientseparation medium (PAA, Colbe, Germany). Isolated CD8 lymphocytes orPBMCs were incubated until use in T-cell medium (TCM) consisting ofRPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10%heat inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100U/ml Penicillin/100 μg/ml Streptomycin (Cambrex, Cologne, Germany), 1 mMsodium pyruvate (CC Pro, Oberdorla, Germany), 20 μg/ml Gentamycin(Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/mlIL-2 (Novartis Pharma, Nümberg, Germany) cytokines were added to the TCMfor this culture step. Isolation of CD8+ lymphocytes was performed bypositive selection using CD8 MicroBeads (Miltenyi Biotec,Bergisch-Gladbach, Germany).

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed as described before (Walter et al. 4974-78) withminor modifications. Briefly, biotinylated peptide-loaded recombinantHLA-A*2402 and HLA-A*0201 molecules lacking the transmembrane domain andbiotinylated at the carboxy terminus of the heavy chain were produced.The purified costimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung,Ledbetter, and Muller-Eberhard 4611-15) was chemically biotinylatedusing Sulfo-N-hydroxysuccinimidobiotin as recommended by themanufacturer (Perbio, Bonn, Germany). Beads used were 5.6 μm largestreptavidin coated polystyrene particles (Bangs Laboratories, Illinois,USA). pMHC used as high and low immunogenic controls were A*0201/MLA-001(peptide ELAGIGILTV (SEQ ID NO:96) from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI (SEQ ID NO:97) from DDX5), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of600 ng biotin-anti-CD28 plus 200 ng relevant biotin-pMHC (high densitybeads). Stimulations were initiated in 96-well plates by co-incubating1×106 CD8+ T cells with 2×105 washed coated beads in 200 μl TCMsupplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37° C., 5%CO2 and 95% relative humidity. Half of the medium was then exchanged byfresh TCM supplemented with 80 U/ml IL-2 and incubation was continuedfor 3-4 days at 37° C. This stimulation cycle was performed for a totalof three times.

Finally, multimer analyses were performed by staining cells withfluorescent A*0201 or A*2402 HLA multimers (produced as described by{Altman, 1996 ALTMAN1996/id}) and CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) or additionally with a viability marker(Live/dead-Aqua or -Violet dye (Invitrogen, Karlsruhe, Germany)), andwere conducted on a four-color FACSCalibur (BD) or a LSRII SORPcytometer (BD; eighteen color, equipped with a blue (488 nm), violet(405 nm), red (640 nm) and green (532 nm), respectively. Peptidespecific cells were calculated as percentage of total CD8+ T cells.Evaluation of multimer analysis was done using the FCSExpress or FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by appropriate gating and bycomparing to negative control stimulations. Immunogenicity for a givenantigen was detected if at least one evaluable in vitro stimulated wellof one healthy donor was found to contain specific CD8+ T-cells after invitro stimulation (i.e. the fraction of multimer+ cell population withinthis well constituted at least 1% of the CD8+ cells, the frequency wasat least 10-fold over the median of the respective negative controls(stimulation with irrelevant and staining with relevant multimer) andthe cells were not located on the diagonal of the plot).

In Vitro Immunogenicity for IMA941 Peptides

For 47 of 54 tested HLA-A*2402 peptides and for 3 of 3 tested HLA-A*0201peptides, in vitro immunogenicity could be demonstrated by generation ofpeptide specific T-cell lines. Exemplary flow cytometry results afterTUMAP-specific multimer staining for two peptides of the invention areshown in FIG. 3 together with a corresponding negative control. Resultsfor 54 A*2402 and 3 A*0201 peptides of the invention are summarized inTable 4.

TABLE 4 In vitro immunogenicity of HLA class I peptides of the inventionResults of in vitro immunogenicity ex-periments conducted by Immatics are showingthe percentage of positive tested donors andwells among evaluable. At least four donorsand 48 wells were evaluable for each peptide. Donors Wells SEQ positive/positive/ ID evaluable evaluable NO: Antigen [%] [%]  1 CDC2-001  83 28 2 ASPM-002  67 32 18 MMP3-001  11  1  4 MET-006  67 21  3 UCHL5-001  7512  7 MST1R-001  50 13 33 KIF2C-001  17  2  9 SMC4-001  73 10 17EPHA2-005   0  0  5 PROM1-001  83 26  6 MMP11-001  33 11  8 NFYB-001  50 7 16 ASPM-001  17  3 20 PLK4-001  60  5 14 ABL1-001  83 18 26 ATAD2-001 33  3 21 ATAD2-002  17  1 27 ATAD2-003   0  0 12 AVL9-001 100 31 22COL12A1-001   0  0 23 COL6A3-001   0  0 24 FANCI-001  17  1 28HSP90B1-001  50  7 15 MUC6-001  83 22 13 NUF2-001 100 50 19 NUF2-002  50 6 11 PPAP2C-001  83 29 25 RPS11-001  17  3 29 SIAH2-001  50  8 30SLC6A6-001  17  1 10 UQCRB-001  83 24 31 IQGAP3-001 100 24 32 ERBB3-001 83 CCDC88A-001   0  0 CCNB1-003  33  3 CCND2-001  17 10 CCNE2-001   0 0 CEA-010  40  3 CLCN3-001  33  6 DNAJC10-001  50 15 DNAJC10-002  33  3EIF2S3-001  17  1 EIF3L-001 100 29 EPPK1-001  17  1 GPR39-001  50  6ITGB4-001  67 20 LCN2-001  17  1 SDHC-001  33  3 PBK-001   0  0POLD3-001  67  7 PSMD14-001  17  1 PTK2-001  17  4 TSPAN1-002  17  1ZNF598-001  83 17

The following peptides were already described in other applications byimmatics and included in the vaccines IMA901 (MET-001 and TOP-001),IMA910 (MET-001 and TOP-001) and IMA950 (IGF2BP3-001). As for exampleMET-001 leads to extremely good in vivo reactions, the data can be seenas an indication for the clinical usefulness of the peptides of theinvention,

SEQ Donors Wells ID positive/ positive/ NO: Antigen evaluable [%]evaluable [%] IGF2BP3-001 50 21 MET-001 67 42 TOP-001 40 10

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The invention claimed is:
 1. A fusion protein comprising a peptideconsisting of the amino acid sequence selected from the group consistingof SYIIDPLNL (SEQ ID NO: 5), VWSDVTPLTF (SEQ ID NO: 6), NYLLYVSNF (SEQID NO: 7), VYTTSYQQI (SEQ ID NO: 8), HYKPTPLYF (SEQ ID NO: 9) andNYEETFPHI (SEQ ID NO: 15) and 80 N-terminal amino acids of an HLA-DRantigen-associated invariant chain (Ii).
 2. A composition comprising apeptide 1 consisting of the amino acid sequence selected from the groupconsisting of SYIIDPLNL (SEQ ID NO: 5), VWSDVTPLTF (SEQ ID NO: 6),NYLLYVSNF (SEQ ID NO: 7), VYTTSYQQI (SEQ ID NO: 8), and HYKPTPLYF (SEQID NO: 9) in the form of a pharmaceutically acceptable salt; and animmunogenicity enhancing amount of at least one adjuvant.
 3. Thecomposition according to claim 2, wherein the pharmaceuticallyacceptable salt is acetic acid or hydrochloric acid.
 4. The compositionaccording to claim 2, wherein the at least one adjuvant is selected fromthe group consisting of imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides andderivates, poly-(I:C) and derivates, RNA, sildenafil, and particulateformations with PLG or virosomes.
 5. The composition according to claim2, wherein the at least one adjuvant is selected from the groupconsisting of a colony-stimulating factor, imiquimod, resiquimod, andinterferon-alpha.
 6. The composition according to claim 2, wherein theat least one adjuvant comprises imiquimod or resiquimod.
 7. Thecomposition of claim 2, wherein said peptide is produced by solid phasepeptide synthesis using a solid-phase support.
 8. The composition ofclaim 7, wherein said peptide is produced by the solid phase peptidesynthesis using a solid-phase support followed by removal from thesolid-phase support by a composition comprising 95% trifluoroacetic acidand a 50% scavenger mix.
 9. The composition of claim 8, wherein thetrifluoroacetic acid is removed by evaporation.
 10. The composition ofclaim 7, wherein the peptide is purified using a method selected fromthe group consisting of re-crystallization, size exclusionchromatography, ion-exchange chromatography, hydrophobic interactionchromatography, and reverse-phase high performance liquidchromatography.
 11. The composition of claim 10, wherein the peptide ispurified using ion-exchange chromatography using an organic or inorganicacid.
 12. The composition of claim 11, wherein the organic acid isselected from the group consisting of acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid,ethane sulfonic acid, p-toluenesulfonic acid, and salicylic acid, andthe inorganic acid is selected from the group consisting of hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid. 13.The composition of claim 12, wherein the organic acid is acetic acid orthe inorganic acid is hydrochloric acid.
 14. The composition of claim10, wherein the peptide is purified using ion-exchange chromatographyusing a base.
 15. The composition of claim 14, wherein the base isselected from the group consisting of sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, and trimethylamine.16. The composition of claim 2, wherein the peptide is produced by amethod comprising yeast cell or bacterial cell expression, wherein thepeptide is subsequently isolated in the form of a pharmaceuticallyacceptable salt after yeast or bacterial cell expression.
 17. Thecomposition of claim 2, further comprising a pharmaceutically acceptablecarrier selected from the group consisting of saline, Ringer's solution,and dextrose solution.
 18. The composition of claim 17, furthercomprising a pharmaceutically acceptable excipient.